Compositions, methods, and systems relating to controlled crystallization and/or nucleation of molecular species

ABSTRACT

The present invention generally relates to compositions, methods, and systems relating to controlled crystallization and/or nucleation of a molecular species. In some embodiments, the crystallization and/or nucleation of the molecular species may be controlled by tuning the surface chemistry and/or the morphology of a crystallization substrate. In some embodiments, the molecular species is a small organic molecule (e.g., pharmaceutically active agent).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/216,018, filed Aug. 23, 2011 entitled “Compositions, Methods, andSystems Relating to Controlled Nucleation of Small Organic Molecules;”which application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/375,925, filed Aug. 23, 2010, and entitled “Compositions,Methods, and Systems Relating to Controlled Nucleation of Small OrganicMolecules;” U.S. Provisional Patent Application Ser. No. 61/418,767,filed Dec. 1, 2010, and entitled “Compositions, Methods, and SystemsRelating to Controlled Nucleation of Small Organic Molecules;” and U.S.Provisional Patent Application Ser. No. 61/466,759, filed Mar. 23, 2011,and entitled “Compositions, Methods, and Systems Relating to ControlledNucleation of Small Organic Molecules,” each of which are incorporatedherein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention generally relates to compositions, methods, andsystems relating to controlled crystallization and/or nucleation ofmolecular species. In some cases, the molecular species is a smallorganic molecule (e.g., a pharmaceutically active agent).

BACKGROUND OF THE INVENTION

In many areas of science and technology, such as the production ofpharmaceuticals, semiconductors, and optics, as well as the formation ofbiominerals, the ability to control crystallization is desired. As willbe known to those of ordinary skill in the art, nucleation is generallya critical step in controlling the crystallization process. In mostembodiments, crystallization starts with heterogeneous nucleation whichoccurs at random foreign surfaces. While many studies have beenconducted regarding controlling crystallization of small organicmolecules, crystallization is a complex and not well understood process.In addition, generally, small organic molecules may be crystallized in avariety of crystal patterns, and it is difficult, if not impossible, topredict under which conditions, a small organic molecule willcrystallize in.

Accordingly, new compositions, methods, and systems are needed.

SUMMARY OF THE INVENTION

In some embodiments, a method of facilitating crystallization isprovided comprising: exposing a substrate comprising pores to amolecular species; and causing molecular species to crystallize in thepresence of at least a portion of the pores with an average inductiontime, wherein the average induction time is decreased by a factor of atleast three, under substantially similar conditions, as compared to theaverage induction time using the substrate substantially free of pores.

In some embodiments, a method of facilitating crystallization isprovided comprising exposing a substrate to a molecular speciescomprising a plurality of functional groups, wherein the substratecomprises a plurality of complimentary functional groups to thefunctional groups of the molecular species on at least one surface ofthe substrate; and causing the molecular species to crystallize on atleast a portion of the substrate with an average induction time, whereinthe average induction time is decreased by a factor of at least three,under substantially similar conditions, as compared to the averageinduction time using a polymeric material not comprising thecomplimentary functional groups.

In some embodiments, a method of facilitating crystallization isprovided comprising exposing a substrate comprising a plurality ofsurface features, to a molecular species wherein the surface featureshave a cross section of at least 10 nm and have a shape and/or angle(s)which is selected to be complimentary to a known shape and/or angle(s)of a selected known crystal structure of the molecular species; andcausing the molecular species to crystallize in at least a portion ofthe surface features, wherein molecular species is substantially formedhaving the selected crystal structure.

In some embodiments, a method of forming a plurality of particlescomprising a crystallized molecular species is provided comprisingproviding a solution containing a plurality of polymeric particleshaving a plurality of pores and a molecular species; and causing themolecular species to crystallize in at least a portion of the pluralityof pores.

In some embodiments, a composition is provided comprising a plurality ofpolymeric particles, wherein the particles comprise pores; and apharmaceutically active agent crystallized in a least a portion of thepores.

In some embodiments, a method of administering a pharmaceutically activeagent to a subject is provided comprising providing a plurality ofpolymeric particles comprising pores and a pharmaceutically active agentcrystallized in at least a portion of the pores; and administering theplurality of polymeric particles to the subject.

In some embodiments, a method of making a pharmaceutical product isprovided comprising crystallizing a pharmaceutically active agent in thepresence of at least one excipient; forming a pharmaceutical productcomprising the pharmaceutically active agent and the at least oneexcipient; wherein the process is free or essentially free of mechanicalsteps for altering the physical properties of the pharmaceuticallyactive agent or the pharmaceutical product.

In some embodiments, a method is provided comprising providing asolution, a substrate comprising pores, and a pharmaceutically activeagent, wherein the pharmaceutically active agent has a greater affinityto the substrate as compared to affinity between the solvent and thepharmaceutically active agent and compared to the affinity between thesolvent and the substrate; and causing the pharmaceutically active agentto crystallize in at least a portion of the pores.

In some embodiments, a method of forming a plurality of particlescomprising a crystallized active agent is provided comprising providinga solution, a substrate comprising a plurality of particles, and apharmaceutically active agent, wherein the pharmaceutically active agenthas a greater affinity to the substrate as compared to the affinitybetween the solvent and the pharmaceutically active agent and theaffinity between the solvent and substrate; and causing thepharmaceutically active agent to crystallize associated with at least aportion of the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates varying interaction of a crystal phase of aspirinwith different substrate functional groups, according to someembodiments.

FIG. 2 provides flow charts of methods for manufacturing pharmaceuticalcompositions, according to known methods and methods of the invention,according to some embodiments.

FIG. 3 shows the nucleation density of aspirin on a variety of substratefilms, according to some embodiments.

FIG. 4A shows cumulative probability distribution of nucleationinduction time and statistical analysis on the same data sets, accordingto some embodiments.

FIG. 4B shows cumulative probability distribution of nucleationinduction time and obtained with polymers synthesized via bulkpolymerization, according to some embodiments.

FIG. 5 shows X-ray diffraction patterns of aspirin crystals nucleatedfrom polymer surfaces and from the bulk, according to some embodiments.

FIGS. 6A-6C show optical micrographs of hydrogel particles, according tosome embodiments.

FIGS. 7 and 8 shows mesh sizes of hydrogel particles measured byequilibrium swelling measurements, according to some embodiments.

FIG. 9 shows an optical microscopy image of ASA crystals on PEG₇₀₀DAparticles, according to some embodiments.

FIG. 10 shows partitioning of aspirin vs. acetaminophen in the PEGDAgel, according to some embodiments.

FIG. 11 shows Hansen parameters, according to some embodiments.

FIG. 12A and FIG. 12B illustrate fabrication methods for polymer filmsusing spherical wells on the surface of a substrate by nanoparticleimprint lithography (NpIL).

FIG. 12C shows AFM images of polyacrylic acid films crosslinked withdivinylbenzene (AA-co-DVB) with and without spherical wells, accordinglyto some embodiments.

FIGS. 13A and 13C shows AFM images of hexagonal and square wells on thesurface a substrate, according to some embodiments.

FIG. 13B shows a TEM image of iron oxide magnetic nanocrystals.

FIG. 13D shows a high resolution SEM image of Si square posts on Siwafer fabricated by AIL for templating square wells on the surface of asubstrate, according to some embodiments.

FIG. 13E shows depth profiles of square and spherical wells of thesurface of a crystallization substrate, according to some embodiments.

FIG. 14 illustrates the effect of the feature shape in AA-co-DVB polymerfilms on the nucleation kinetics of aspirin, according to someembodiments.

FIG. 15A shows an AFM phase image of aspirin crystals grown in squarewells on the surface of a substrate, according to some embodiments.

FIG. 15B shows an AFM phase image showing (100) layers of aspirincrystals nucleated at ledges in the square wells on the surface of asubstrate, according to some embodiments.

FIG. 15C and FIG. 15D illustrate representative epitaxy configurationsof aspirin crystal facets along the ledge of a square wells on thesurface of a substrate, according to some embodiments.

FIG. 15E shows proposed aspirin-polymer interactions at acrystal-polymer interface, according to some embodiments.

FIG. 15F shows a proposed epitaxy configuration of aspirin crystalfacets at the corner of hexagonal wells on the surface of a substrate,according to some embodiments.

FIG. 15G and FIG. 15H show AFM phase images of an aspirin crystallitegrown from hexagonal wells on the surface of a substrate and possibleorientations, according to some embodiments.

FIG. 15I shows an AFM height image of the surface of an aspirin crystalgrown on and detached from an AA-co-DVB polymer film having hexagonalwells on the surface of a substrate, according to some embodiments.

FIG. 16 illustrates the effect of polymer surface chemistry on thekinetics of angular features-induced nucleation of aspirin, according tosome embodiments.

FIG. 17 shows X-ray diffraction patterns of aspirin crystals grown froma butyl acetate bulk solution (top), on substrates having square wellson the surface of a substrate (middle), and hexagonal wells on thesurface of a substrate (bottom), according to some embodiments.

FIG. 18 shows a statistical analysis of aspirin nucleation inductiontimes with and without wells on the surface of a substrate, according tosome embodiments.

FIG. 19 shows mesh sizes of polymer hydrogels estimated by equilibriumswelling measurements and SANS analysis, according to some embodiments.

FIG. 20 shows absolute SANS intensity spectra for polymer hydrogels,according to some embodiments.

FIG. 21 shows schematics of microgel structures inferred from SANSmeasurements, according to some embodiments.

FIG. 22 graphs a comparison of partition coefficient, κ, in hydrogelsfor ASA (top) and ACM (bottom), according to some embodiments.

FIG. 23 shows enthalpy isotherms for adsorption of ASA onto hydrogels,including instantaneous (top) and cumulative (bottom) enthalpies ofadsorption, according to some embodiments.

FIG. 24A graphs the effect of polymer mesh sizes on nucleation kinetics,according to some embodiments.

FIG. 24B and FIG. 24C graphs comparison of two exponential vs. stretchedexponential models using PEG₅₇₅DA-co-AM (b) and PEG₇₀₀DA-co-AM (c) asrepresentative examples.

FIG. 25A shows XRD patterns of ASA crystals grown from PEGDA polymerfilms, PEGDA-co-AM polymer films, and from a bulk solution, according tosome embodiments.

FIGS. 25B-25E show optical images of ASA crystals nucleated from bulk(FIG. 25B), a PEGDA-co-AM surface (FIG. 25C), and a PEGDA surface (FIGS.25D-25E), according to some embodiments.

FIG. 25F and FIG. 25G illustrate molecular structures of (002) and (011)facets of ASA crystals, according to some embodiments.

FIG. 26A shows XRD patterns of ACM crystals grown from PEGDA films, andPEGDA-co-AM polymer films, and bulk solution, according to someembodiments.

FIGS. 26B-26D show optical images of ACM crystals nucleated from bulk(FIG. 26B), a PEGDA surface (FIG. 26C), and a PEGDA-co-AM surface (FIG.26D), according to some embodiments.

FIG. 26E shows the ACM molecular structure.

FIGS. 26F-26H show the molecular structures of the (011), (022), (111)and (101) facets of ASA crystals.

FIG. 27 shows optical micrographs of PEG400

DA microgels as synthesized (left) and CBZ form II needles grown onPEG400DA (right), according to some embodiments.

FIG. 28 illustrates the effect of solute concentration on thepolymorphic composition of CBZ crystals, according to some embodiments.

FIGS. 29A-29E show CBZ-polymer interactions inferred from preferredcrystal orientations, according to some embodiments.

FIGS. 30A-30I show optical micrographs of ROY crystallized from (FIGS.30A-30C) bulk and on (FIGS. 30D-30I) microgels, according to someembodiments.

FIG. 31 shows polymorph frequency of occurrence in 12 mg/ml ROY-ethanolsolution with or without microgels of various mesh sizes (left) and athigher solution concentrations (right), according to some embodiments.

FIGS. 32A-32D show specific polymer-ROY interactions inferred frompreferred crystal orientations, according to some embodiments.

FIGS. 33A-33C show schematics illustrating the mesh size effect onnucleation, according to some embodiments.

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION

The present invention generally relates to compositions, methods, andsystems relating to the controlled crystallization and/or nucleation ofmolecular species (e.g., small organic molecules). The subject matter ofthe present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

According to some aspects of the present invention, compositions,methods, and systems are provided for controlling the nucleation and/orcrystallization of molecular species, e.g. from a solution by tuning thesurface chemistry and/or morphology of crystallization substrates. Thedescription provided herein describes how to select and/or vary acrystallization substrate (e.g., a material on which a molecular speciesis to crystallize) to affect the manner in which a molecular speciescrystallizes. The present invention recognizes that appropriateselection of the type of crystallization substrate (e.g., the chemicalmake-up of the substrate) as well as the morphology of thecrystallization substrate (e.g., surface topology, porosity, etc.) usedto promote nucleation of a small organic molecule can lead improvecrystallization kinetics (e.g., reduced induction time) and/orcrystallization of the molecular species in a selected crystal form. Themethods and/or systems described herein can be used affect thecrystallization rate and/or the type of crystal structure formed (e.g.,the polymorph). For example, in some embodiments, the present inventionrecognizes that selection of the substrate as well as the porosity ofthe substrate used for nucleation of a small organic molecule can leadto a reduced average induction time as compared to a similar substratethat does have pores, under substantially similar conditions.

It should be understood that while many of the embodiments describedherein discusses the use of a molecular species being a small organicmolecule, this is by no way limiting and other molecular species may beemployed (e.g., inorganic salts).

It is a feature of the invention that average crystallization inductiontimes can be decreased significantly in all of the arrangementsdescribed herein for improved crystallization, for example, utilizing aporous substrate as compared to a non-porous substrate, utilizing asubstrate with complimentary functional groups, utilizing a substratewith surface features complimentary to morphological features (shape,angle, etc.) of known crystal structures, etc., as compared tosubstrates not having those features. In all embodiments herein, oneembodiment involves reducing the average induction time by a factor ofat least 3 (or other factors described herein), under substantialidentical conditions, as compared to the average induction time using asubstrate substantially free of one or more (or all) of featuresdescribed.

Selection, control, and/or modification of the crystallization substratematerial (e.g., which forms the crystallization substrate) will now bedescribed in more detail. In some embodiments, the substrate comprises apolymeric material. The polymeric material may form a hydrogel. In somecases, the polymeric material is porous. The polymeric material may alsobe formed such that at least one surface of the polymeric materialcomprises surface features to aid in the crystallization and/ornucleation processes. It should be understood, that while much of thediscussion provided herein focuses on crystallization substratescomprising a polymer material (e.g., a hydrogel), this is by no meanslimited and other materials may be employed as crystallizationsubstrate, providing the material is capable of comprising a selectedsurface chemistry and/or morphology (e.g., inner surface and outersurface morphologies).

As noted above, the crystallization and/or nucleation of a small organicmolecule may be affected by the surface chemistry and/or morphology of acrystallization substrate. This discussion first focuses on the surfacechemistry of the crystallization substrate. In some embodiments, thecrystallization substrate material may be selected such that thesurface(s) of the crystallization substrate material (e.g., outersurfaces and/or the surface of the pores, if present) comprises aplurality of at least one type of functional group which iscomplimentary to at least one functional group of the small organicmolecule. That is, the functional groups of the substrate may beselected so as interact with a specific functional group of the organicsmall molecule of interest. The selection of complimentary combinationsof functional groups can results in crystallization of the small organicmolecule in 1) a particular crystal form (e.g., polymorph) and/or 2)with a faster induction time.

Without wishing to be bound by theory, selection of complimentaryfunctional groups for the substrate (e.g. as a feature of the substratematerial itself, and/or a surface coating and/or pattern of species onthe substrate) may result in the formation of a particular crystal formof the small organic molecule due the preferential interactions betweenthe portion of the small organic molecule having the function group andthe substrate. The preferential interactions may cause the portion ofthe small organic molecule having the functional group to have a greateraffinity for the surface of the substrate as compared to the otherportions of the small organic molecule, thereby causing a plurality ofthe small organic molecules to associate with the substrate in a favoreddirectional orientation. The alignment of a plurality of small organicmolecules having the same approximate directional orientation cancatalyze the nucleation/crystallization of the small organic molecule ina particular crystal phase. In embodiments where the crystalstructure/form of the small molecule is known, the crystalstructure/form can provide a basis for selecting the type ofcomplimentary functional groups to be present on the surface of thecrystallization substrate. That is, a known crystal structure may beexamined to determine which functional groups of the small organicmolecule are present on the surface of at least one face/edge of thecrystal form and a type of functional group complimentary to that can beselected to be present of the substrate surface.

For example, as described in more detail in Example 1, a known polymorphof aspirin is associated with a substrate via different faces of thecrystal depending on the functional groups on the surface of thesubstrate. As shown in FIG. 1, three different types of functionalgroups (e.g., —COOH, —COMe, and phenyl) are concentrated on threedifferent faces of the crystal. Substrates were selected to comprise aplurality of complimentary functional groups to the three types offunctional group present on each of the three crystal faces, and thisresulted in aspirin crystallizing and associating with each of thesubstrates via different crystal faces. Thus, for molecular species withknown crystal structures, the substrate can be selected comprising aplurality of functional groups on the surface which are selected to becomplimentary to a functional group on/at the edge/face of the knowncrystal form of the molecular species. In embodiments where a crystalstructure of the molecular species is not known (e.g., for a molecularspecies which has not been crystallized previously and/or for which acrystal structure has not yet been obtain), those of ordinary skill inthe art will be able to select crystallization substrates withfunctional groups which are complimentary to the functional groupspresent for the molecular species, which may result in the formation ofa variety of types of crystals. In addition, as described herein, theaffinity for the small molecule for the substrate may also result inlocal regions of supersaturation.

The selection of functional groups on the surface of the substrate mayalso be employed to improve the induction time for the crystallizationof the molecular species. In such cases, the affinity for the molecularspecies to associate with the substrate may draw the molecular speciestowards the substrate, and hence increases the chances of nucleation,thus reducing the nucleation time. In some cases, the greaterinteraction between the small organic molecule and the substrate isembodied by higher concentrations of the small organic molecule in thepore of a substrate and/or near a surface of the substrate as comparedto that in the bulk phase, for example, as illustrated by FIG. 13. Thatis, local area(s) of supersaturation may be present at the surfaces(e.g., outer surface and surface of the pores, if present) of thesubstrate.

In addition, in some embodiments of the present invention, the solvent,the crystallization substrate, and the small organic molecule may beselected such that the small organic molecule has a strongerinteraction/affinity with the crystallization substrate as compared tothe solvent. The rate of crystallization and/or nucleation may beincreased in embodiments where the small organic molecule has preferredinteractions with the crystallization substrate over the solvent, ascompared to embodiments where there is no preferential interactions. Inaddition, the interaction/affinity between the substrate and the solventmay be greater than the interaction/affinity between the solvent and thesubstrate. Without wishing to be bound by theory, a greater interactionof the small organic molecule with the substrate as compared to any ofthe other interactions in the system (e.g., between the small organicmolecule and the solvent, between the substrate and the solvent) may aidin reducing the average induction time, as the small organic molecule isdrawn towards the substrate, and hence increases the chances ofnucleation. Those of ordinary skill in the art will be capable ofselecting combinations of solvents and crystallization substratematerials for a selected small organic molecule, based on the teachingdescribed herein, which have the desired affinities/interactions betweenthe solvent, the small organic molecule, and the crystallizationsubstrate. In some embodiments, determining the concentration of a smallorganic molecule in the pores of a porous substrate may aid indetermining an optimal substrate/solvent/small organic moleculecombination.

Complimentary types functional groups (e.g., comprised on the surface ofthe substrate and the molecular species) will be known to those ofordinary skill in the art. The association may be based on formation ofa bond, such as an ionic bond, a covalent bond (e.g., carbon-carbon,carbon-oxygen, oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen,carbon-nitrogen, metal-oxygen, or other covalent bonds), a hydrogen bond(e.g., between hydroxyl, amine, carboxyl, thiol, and/or similarfunctional groups), a dative bond (e.g., complexation or chelationbetween metal ions and monodentate or multidentate ligands), Van derWaals interactions, or the like.

In some cases, the crystallization substrate material may be selectedsuch that it comprises at least a plurality of hydroxyl functionalgroups, a plurality of carboxylic acid ester functional groups, aplurality of nitrogen containing base functional group, a plurality ofaryl (e.g., phenyl) functional groups, a plurality of carboxylfunctional group, a plurality of tertiary amide functional groups, orcombinations thereof. As a non-limiting example, if the small organicmolecule comprises an aryl group, the functional group on the surface ofthe substrate may be selected to be an aryl functional group, such thatpi-interactions can occur between the surface of the substrate and thesmall organic molecule. As another example, if the small organicmolecule comprises a hydrogen-bond donating group, the functional groupon the surface of the substrate may be selected to be a hydrogen-bondaccepting group. As a specific example, the small organic molecule maycontain a carboxylic acid functionality and the surface of the substratemay contain a tertiary amide functionality. As another specific example,the small organic molecule may contain a carbonyl group and the surfaceof the substrate may contain a hydroxyl group. As yet another specificexample, both the small organic molecule and the surface of thesubstrate may contain phenyl groups, and the interaction may be api-stacking interaction.

Those of ordinary skill in the art will be aware of methods of formingmaterial comprising a plurality of a selected type of functional group.In some embodiments, the substrate comprises a polymer material, whereina portion of the monomeric units forming the polymer each comprise aselected type of functional group. For example, the monomers shown belowmay be polymerized to form a polymer comprising the functional groups ofthe monomers:

In some embodiments, in addition to selecting a crystallizationsubstrate based on the surface chemistry, the morphology of thecrystallization substrate can also be varied to affect thecrystallization and/or nucleation of a molecular species (e.g., smallorganic molecule). The morphology of a crystallization substrate may bevaried by changing 1) the outer surface morphology (e.g., features suchas wells) and/or 2) the inner surface morphology (e.g., such that thecrystallization substrate is porous). While much of the discussionherein focuses on embodiments wherein only the outer surface morphologyor the inner surface morphology is selected and/or optimized, this is byno means limiting, and those of ordinary skill in the art will be ableto apply the teachings herein to embodiments where both the outersurface and the inner surface morphology are selected and/or optimized.

As described in more detail below, in some cases, the substrate maycomprise a plurality of pores, wherein the small organic molecule maycrystallize in at least some of the pores and/or the surface of thesubstrate may comprise a plurality of features (e.g., wells) wherein thesmall organic molecule may crystallize in at least a plurality of thepores and/or features. The pore and/or feature size and/or shape may beselected so as to increase the rate of crystallization (e.g., reducedinduction time) and/or to promote crystallization of the small organicmolecule is a selected crystal form.

Selection of the outer surface morphology will now be described in moredetail. In some embodiments, the outer surface morphology of thecrystallization substrate may be selected so as to promotecrystallization (e.g., by increasing the induction rate and/or bypromoting the formation of a certain crystal form) of a selected crystalform of a small organic molecule. At least one outer surface of thesubstrate may comprise a plurality of features having a shape which iscomplimentary to a known crystal form (e.g., polymorph) of the smallorganic molecule. For example, if a crystal form is known for a smallorganic molecule, the shape and/or angle(s) of the crystals are known orcan be deduced/calculated. Based at least in part on the knowledge ofthe shape and/or angle(s) of the crystals, a complimentary shape and/orangle(s) of a plurality of features formed in the surface of thecrystallization substrate may be selected. Without wishing to be boundby theory, selection of a complimentary shape and/or angle(s) of thefeatures may promote the grown of the crystals as the features candirect the nucleation in a minimal-stress configuration due to ageometrical match between the crystal form and the features. As anon-limiting example, for a crystal form which is known to have an angleof approximately 120°, a hexagonal well on the surface of a substratemay promote the growth of that crystal form. As another non-limitingexample, for a crystal form which is known to have an angle ofapproximately 90°, a square or well on the surface of a substrate maypromote of the growth of that crystal form. As yet another non-limitingexample, for a crystal form which is known to have an angle ofapproximately 60°, a triangular well on the surface of a substrate maypromote the growth of that crystal form.

In some embodiments, the features formed on at least one surface of thesubstrate comprise a plurality of wells. The features formed in theouter surface of the crystallization substrate may be of any suitableshape and size. In some cases, each of the feature may have the shape ofa circle, an oval, a triangle, a square, a rectangle, a trapezoid, apentagon, a hexagon, an octagon, etc. In some cases, the shape does notcomprise round edges. In some cases, the shape is not a circle or anoval. Generally, the features formed in the outer surface of thecrystallization substrate have a cross section of at least 10 nm. Insome cases, the cross section length is between about 10 nm and about1000 nm, between about 10 nm and about 900 nm, between about 10 nm andabout 800 nm, between about 10 nm and about 700 nm, between about 10 nmand about 600 nm, between about 10 nm and about 500 nm, between about 10nm and about 400 nm, between about 10 nm and about 300 nm, between about10 nm and about 200 nm, between about 10 nm and about 100 nm, betweenabout 50 nm and about 500 nm, between about 100 nm and about 500 nm, orany range therein. In some cases, the features have an average depth ofless than about 10 mm, or less than about 5 mm, or less than about 1 mm,less than about 500 um (micrometer), or less than about 100 um, or lessthan about 50 um, or less than about 1000 nm, or less than about 500 nm,or less than about 100 nm, or less than about 50 nm, or less than about40 nm, or less than about 30 nm, or less than about 20 nm, or less thanabout 10 nm, or less than about 5 nm. In some cases, the features havean average depth of between about 10 mm and about 1 nm, between about 10mm and about 100 nm, between about 10 mm and about 1 mm, between about 5mm and about 1 mm, between about 5 mm and about 1 um, between about 50nm and about 1 nm, between about 40 nm and about 1 nm, between about 30nm and about 1 nm, between about 20 nm and about 1 nm, between about 10nm and about 1 nm, or between about 10 nm and about 5 nm. Thecrystallization substrate may comprise any suitable number of features,for example, at least about or about 5 features, 10 features, 20features, 50 features, 100 features, 200 features, 500 features, 1000features, 2000 features, 5000 features, 10,000 features, 50,000features, or more. In some cases, the features are formed in a singlesurface of the crystallization substrate. In some cases, the featuresare formed in more than one surface of the crystallization substrate.The size and/or shape of the crystallization substrate itself may beselected depending on the desired number, size, and/or shape of thefeatures. Those or ordinary skill in the art will be aware of methodsand systems for determining the size, shape, and/or number of features.Methods for forming suitable substrates comprising surface features willalso be known to those of ordinary skill in the art and are describedherein. In some embodiments, a substrate comprising a plurality ofsurface features is not porous.

Selection of the inner surface morphology will now be described in moredetail. In some embodiments, the inner surface morphology of thecrystallization substrate may be selected so as to promotecrystallization of a selected small organic molecule (e.g., byincreasing the crystallization kinetics). In some cases, acrystallization substrate may be porous (e.g., in addition to anysurface wells). In some cases, a method comprises exposing a poroussubstrate to a small organic molecule and causing the small organicmolecule to crystallize in at least a portion of the pores with areduced induction time as compared to using the same substrate nothaving any pores under essential identical conditions (e.g., using thesame or a substantially similar temperature, solvent(s), container(s),concentration of the molecular species, substrate shape, substrate size,substrate material, etc.). That is, the present invention recognizesthat the porosity of the substrate used for nucleation of a smallorganic molecule can lead to a reduced average induction time ascompared to a similar substrate that does not have pores. The termaverage induction time is given its ordinary meaning in the art andgenerally refers to the time elapsed prior to the formation of adetectable amount of a crystalline phase.

In some cases, the small organic molecule is crystallized in at least aportion of the pores of the substrate with an average induction timewhich is at least 2 times, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, at least 10 times, at least 15 times, at least 20 times, atleast 25 times, at least 50 times, at least 100, or greater, less thanthe average induction time using the substrate not having any pores,under essentially identical conditions. In some cases, the small organicmolecule is crystallized in at least a portion of the pores of thesubstrate with an average induction time which is between about twotimes and about 1000 times, between about 2 times and about 100 times,between about 2 times and about 50 times, between about 2 times andabout 40 times, between about 2 times and about 30 times, between about2 times and about 10 times, etc., less than the average induction timeusing the substrate not having any pores, under essentially identicalconditions. For example, according to the invention the averageinduction time for aspirin using a porous and non-porous substrateformed of 4-acryloylmorpholine polymer is, in one set of commonconditions, 8.8 and 38.2 hours, respectively, and for a porous andnon-porous substrate formed of 4-hydroxybutyl acrylate polymer are 54.6and 243.9 hours, respectively. In some cases, the average induction timeis the average of about 10, about 20, about 30, about 40, about 50,about 60, about 70, about 80, about 90, about 100, about 150, about 200,about 500, or more, experiment conducted under substantially similarconditions.

The pores formed in a crystallization substrate may comprise a range ofsizes and/or be substantially uniform in size. In some cases, the poresmay or might not be visible using imaging techniques (e.g., scanningelectron microscope). The pores may be open and/or closed pores. In somecases, the substrate comprises pores having dimensions from 1-10nanometers, and/or from 10-1000 nanometers and/or from 1-100 microns. Insome embodiments, the average pore size is less than about 10 nm. Insome embodiments, the average pore size is between about 1 nm and about2 nm, between about 1 nm and about 5 nm, between about 0.1 nm and about10 nm, between about 0.01 nm and about 10 nm, between about 0.5 nm andabout 2.5 nm, or between about 1 nm and 20 nm. In some cases, theaverage pore size is about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm,about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm,about 9 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, orabout 200 nm. In some cases, the pore size may be selected so as tofacilitate the crystallization (e.g., as described in the examples).Those or ordinary skill in the art will be aware of methods and systemsfor determining the size of the pores. Methods for forming suitablesubstrates comprising features will also be known to those of ordinaryskill in the art and are described herein. In some cases, the substratecomprising a plurality of pores is provided as a plurality of particles.That is, the substrate comprises a plurality of porous particles. Thesize of the pores may be based on a variety of methods, includingpolymerizing a material in a plurality of substantially similarmaterials comprising different pore sizes (or mesh sized). In somecases, the optimal pore size may be estimated based on the criticalnucleus size (e.g., see Examples).

In some embodiments, wherein the crystallization substrate comprises ahydrogel, the pore size may be better defined as a mesh size. In someembodiments, the average mesh size is less than about 10 nm. In someembodiments, the average mesh size is between about 1 nm and about 2 nm,between about 1 nm and about 5 nm, between about 0.1 nm and about 10 nm,between about 0.01 nm and about 10 nm, between about 0.5 nm and about2.5 nm, or between about 1 nm and 20 nm. In some cases, the average meshsize is about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm, about 3 nm,about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm,about 10 nm, about 20 nm, about 50 nm, about 100 nm, or about 200 nm.Those of ordinary skill in the art will be aware of suitable methods andtechniques for determining the mesh size of a hydrogel, including, butno limited to, small angle neutron scattering (SANS) measurements andequilibrium swelling measurements (e.g., see description of measurementsdescribed in Example 4).

SANS techniques and methods will be known to those of ordinary skill inthe art. In some cases, SANS comprises forming a hydrogel in ascattering cell (e.g., by filling the cell with hydrogel precursors andinitiating polymerization in the cell (e.g., by exposing the cell to UVirradiation)) or providing the hydrogel to the scattering cell. In somecases, the path length through the cell is about 1 mm. SANS instrumentsare commercially available. In some cases, the cell may be maintained atabout 25° C. and the samples may be equilibrized in this atmosphere forat least 30 minutes prior to the measurement. Scattering using incidentneutrons of wavelength λ=6 Å and a wavelength spread (FWHM) of Δλ/λ=11%can be collected at detector distances of 1 m with 20 cm offset, 4 m,and 13.5 m for high-q measurements. Scattering using incident neutronsof wavelength λ=8.09 Å and a wavelength spread (FWHM) of Δλ/λ=11% can becollected at a detector distances of 15.3 m for low-q measurements.USANS measurements were performed on the BT5 perfect crystaldiffractometer within the 6CB sample environment. Data may be reducedusing a commercially available software program (e.g., NIST IGOR) todetermine the mesh size. Further details of suitable calculations aredescribed in Example 4.

The crystallization substrate may be of any suitable shape, size, orform. In some cases, the substrate may be a planar surface and/or aportion of a container. Non-limiting examples of shapes include sheets,cubes, cylinders, hollow tubes, spheres, and the like. In some cases,the maximum dimension of the substrate in one dimension may be at leastabout 1 mm, at least about 1 cm, at least about 5 cm, at least about 10cm, at least about 1 m, at least about 2 m, or greater. In some cases,the minimum dimension of the substrate in one dimension may be less thanabout 50 cm, less than about 10 cm, less than about 5 cm, less thanabout 1 cm, less than about 10 mm, less than about 1 mm, less than about1 um, less than about 100 nm, less than about 10 nm, less than about 1nm, or less.

In some cases, the substrate may comprise a plurality of particles(e.g., polymeric particles). In some cases, a particle may be ananoparticle, i.e., the particle has a characteristic dimension of lessthan about 1 micrometer, where the characteristic dimension of aparticle is the diameter of a perfect sphere having the same volume asthe particle. The plurality of particles, in some embodiments, may becharacterized by an average diameter (e.g., the average diameter for theplurality of particles). In some embodiments, the diameter of theparticles may have a Gaussian-type distribution. In some cases, theplurality of particles may have an average diameter of less than aboutan average diameter of less than about 5 mm, or less than about 4 mm, orless than about 3 mm, or less than about 2 mm, or less than about 1 mm,or less than about 500 um, or less than about 100 um, or less than about50 um, or less than about 10 um, or less than about 1 um, or less thanabout 800 nm, or less than about 500 nm, or less than about 300 nm, orless than about 250 nm, or less than about 200 nm, or less than about150 nm, or less than about 100 nm, or less than about 50 nm, or lessthan about 30 nm, or less than about 10 nm, or less than about 3 nm, orless than about 1 nm, in some cases. In some embodiments, the particlesmay have an average diameter of at least about 5 nm, at least about 10nm, at least about 30 nm, at least about 50 nm, at least about 100 nm,at least about 200 nm, at least about 500 nm, at least about 800 nm, atleast about 1000 nm, at least about 10 um, at least about 50 um, atleast about 100 um, at least about 500 um, at least about 1 mm, at leastabout 2 mm, at least about 3 mm, at least about 4 mm, at least about 5mm, or greater. In some cases, the plurality of the particles have anaverage diameter of about 10 nm, about 25 nm, about 50 nm, about 100 nm,about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500 nm,about 800 nm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5mm, or greater.

Those of ordinary skill in the art will be aware of methods of formingmaterials having the desired surface chemistries and morphologiesdepending of the selected application and small organic molecule.

In some embodiments, the crystallization substrate comprises a polymer.Polymers generally are extended molecular structures comprisingbackbones which optionally contain pendant side groups, wherein the termbackbone is given its ordinary meaning as used in the art, e.g., alinear chain of atoms within the polymer molecule by which other chainsmay be regarded as being pendant. Typically, but not always, thebackbone is the longest chain of atoms within the polymer. A polymer maybe a co-polymer, for example, a block, alternating, or randomco-polymer. A polymer may also comprise a mixture of polymers. In someembodiments, the polymer may be acyclic or cyclic. A polymer may becrosslinked, for example through covalent bonds, ionic bonds,hydrophobic bonds, and/or metal binding. A polymer may be obtained fromnatural sources or be created synthetically.

An exemplary, non-limiting list of polymers that are potentiallysuitable for use in the invention includes polysaccharides;polynucleotides; polypeptides; peptide nucleic acids; polyurethane;polyamides; polycarbonates; polyanhydrides; polydioxanone;polyacetylenes and polydiacetylenes; polyphosphazenes; polysiloxanes;polyolefins; polyamines; polyesters; polyethers; poly(ether ketones);poly(alkaline oxides); poly(ethylene terephthalate); poly(methylmethacrylate); polystyrene; poly(lactic acid)/polylactide; poly(glycolicacid); poly(lactic-co-glycolic acid); poly(caprolactone);polysaccharides such as starch; poly(orthoesters); poly(anhydrides);poly(ether esters) such as polydioxanone; poly(carbonates); poly(aminocarbonates); and poly(hydroxyalkanoates) such as poly(3-hydroxybutyrate)and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and derivatives andblock, random, radial, linear, or teleblock copolymers, cross-linkablematerials such as proteinaceous materials and/or blends of the above.Also suitable are polymers formed from monomeric alkylacrylates,alkylmethacrylates, alpha-methylstyrene, vinyl chloride and otherhalogen-containing monomers, maleic anhydride, acrylic acid,acrylonitrile, and the like. Monomers can be used alone, or mixtures ofdifferent monomers can be used to form homopolymers and copolymers.Other potentially suitable polymers are described in the PolymerHandbook, Fourth Ed. Brandrup, J. Immergut, E. H., Grulke, E. A., Eds.,Wiley-Interscience: 2003, which is incorporated herein by reference inits entirety.

The polymer may have any suitable molecular weight. For example, in someembodiments, the polymer may have an average molecular weight greaterthan 1000 Da, in certain embodiments greater than 5000 Da, in certainembodiments greater than 10000 Da, in certain embodiments greater than20000 Da, in certain embodiments greater than 50000 Da, in certainembodiments greater than 100000 Da, in certain embodiments greater than500000 Da, or in certain embodiments greater than 1000000 Da. In someembodiments, the polymer may have at least 5 subunits, in certainembodiments at least 10 subunits, in certain embodiments at least 20subunits, in certain embodiments at least 30 subunits, in certainembodiments at least 50 subunits, in certain embodiments at least 100subunits, in certain embodiments at least 500 subunits, in certainembodiments at least 1000 subunits, or in certain embodiments at least5000 subunits.

In some embodiments, a polymer may be biodegradable. In otherembodiments, a polymer may be nondegradable. In embodiments where theparticles are to be comprised in a composition for administration to asubject, the polymeric materials may be non-toxic and/or bioabsorbable.

In some cases, the polymeric material may form a hydrogel. As usedherein, the term hydrogel is given its ordinary meaning as used in theart, e.g., a network of polymer chains in an aqueous dispersion medium.In some embodiments, a hydrogel may comprise a plurality of crosslinkedpolymer chains. In some cases, a hydrogel is formed by crosslinking thepolymer chains. Non-limiting examples of polymers capable of forminghydrogels include, silicon-containing polymers, polyacrylamides,crosslinked polymers (e.g., polyethylene oxide, polyAMPS andpolyvinylpyrrolidone), polyvinyl alcohol, acrylate polymers (e.g.,sodium polyacrylate), and copolymers with an abundance of hydrophilicgroups. In some cases, the polymeric materials may be an organogel,wherein the polymer network may be swollen by addition of an organicsolvent. In some cases, the crystallization substrate comprising aplurality of porous hydrogel particles.

In some embodiments, the polymeric material may form a gel. As usedherein, the term gel is given its ordinary meaning in the art and refersto polymer chains that may be crosslinked to form a network, wherein thenetwork may be able to trap and contain fluids. Depending on the levelof crosslinking, various properties of a particular gel can be tailored.For example, a highly crosslinked gel may generally be structurallystrong and may resist releasing fluid under pressure. Those of ordinaryskill in the art would be able to identify methods for modulating thedegree of crosslinking in such gels.

Non-limiting examples of monomers which may be used for preparingpolymeric materials for use with the invention are shown in Scheme 1.Scheme 1 also indicates possible functional groups. It should beunderstood that the monomers shown here are non-limiting, and those ofordinary skill in the art will be able to select other appropriatemonomers and/or polymers for use with the invention.

Those of ordinary skill in the art will be aware of a variety of methodsfor forming polymeric materials having a selected size and/or shape. Insome embodiments, a polymeric material may be formed using UVpolymerization. In some embodiments, a polymeric material may be formedby reacting at least one monomer and a crosslinking agent using UVirradiation. That is, the polymeric films may be synthesized byultraviolet curing a mixture of one or more monomers and optionally acrosslinker (e.g., ethylene glycol dimethacrylate (EGDMA)). In somecases, a porous polymeric material (e.g., particles) can be formed usingphotopolmerization induce phase separation, polymerization with highmolecular weight polymers or nanoemulsions as the porogen, chemicalcrosslinking, catalytic polymerization, etc.

In a particular embodiment, a polymer material may be formed byphotopolymerization induced phase separation. Generally, a mixture of atleast one monomer and a crosslinker are provided in a solvent. Uponexposure to electromagnetic radiation (e.g., UV irradiation), thepolymerization of the monomer and reaction with the crosslinker occurs.As polymer chains reach a critical length, the dynamic asymmetry betweenthe solvent molecules and the polymer cause local regions of highsolvent concentration, which forms the pores. Removal of the solvent(e.g., via evaporation) leaves a porous polymer material.

Those of ordinary skill in the art will be aware of methods andtechniques for forming polymeric materials comprising a desired outersurface morphology, including, but not limited to nanoimprintlithography, nanosphere lithography, and stop flow lithography. In someembodiments, the polymeric materials may be formed using a techniquestermed herein as “nanoparticle imprint lithography” or NpIL. Asdescribed in more detail in Example 3, NpIL comprises providing asubstrate and a plurality of particles and covalently linking (orotherwise immobilizing, e.g., via other strong bonding interactions,adhesion, etc.) the plurality of particles to the surface of a substrateto provide a lithography template. In some cases, this may beaccomplished by crosslinking a functional group present on the surfaceof the substrate with a functional group on the particle. The particlesmay be associated with the substrate surface such that only a singlelayer of the particles is present on the substrate surface. This may beaccomplished, in some embodiments, by selecting surface functionalitiesthat are of such a length that they could not associate with a secondlayer of particles. Following association of the particles with thesurface, any excess particles which do not become associated with thesubstrate can be removed (e.g., by washing the lithography template).General lithography techniques can then be carried out using thelithography template. For example, the lithograph template can beexposed to a polymeric precursor, wherein the polymeric precursor iscured (e.g., via exposure to UV irradiation) following the exposure.Thus, the polymeric material formed comprises an imprint of thelithography template. The polymeric material and the lithographytemplate can then be separated (e.g., by physical manipulation), thusforming a crystallization substrate comprising a plurality of features.Other non-limiting techniques for forming features in the surface of amaterial comprise photofabrication, etching, electrodischarge machining,electrochemical machining, laser beam machining, wire electricaldischarge grinding, focused ion beam machining, micromilling,micro-ultrasonic machining, and micropunching.

As noted above, while much of the discussion focuses on crystallizationsubstrates comprising polymeric materials, this is by no ways limitingand a crystallization substrate may comprise non-polymeric materials.For example, in some embodiments the substrate may comprise a metal, analloy, an inorganic material, etc. wherein the surface of the materialcomprises or is optionally functionalized with a plurality of functionalgroups, and wherein the material may be shaped and/or formed having thedesired inner and/or outer morphologies. It should be understood thatthe functional groups may be a portion of the material, or mayoptionally be formed on the material (e.g., as a coating). For example,a material (e.g., polymeric or otherwise) may form a basis (e.g., core)of the substrate and the surfaces of the substrate may be associatedwith (e.g., coated) with a polymeric material as described hereincomprising a plurality of functional groups.

Crystallization of molecular species (e.g., small organic molecules) maybe carried out according to methods known to those of ordinary skill inthe art. In some cases, a substrate (e.g., as described herein) may beexposed to a solution comprising a small organic molecule. Generally,the small organic molecule is substantially soluble in the solventselected. In some cases, the solution comprising the solvent and thesmall organic molecule may be filtered prior to exposing the solution tothe substrate. The small organic molecule may be present in the solventat a concentration of about 0.05 M, about 0.1 M, about 0.2 M, about 0.3M, about 0.4 M, about 0.5 M, about 0.75 M, about 1 M, about 2 M, orgreater. Non-limiting examples of solvents include water, acetone,ethanol, acetonitrile, benzene, p-cresol, toluene, xylene, mesitylene,diethyl ether, glycol, petroleum ether, hexane, cyclohexane, pentane,dichloromethane (methylene chloride), chloroform, carbon tetrachloride,dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide,hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine,picoline, and combinations thereof.

Those of ordinary skill in the art will be aware of methods for inducingcrystallization. For examples, in some cases, a system comprising asubstrate and a solution comprising the small organic molecule may becooled. Alternatively, the solution comprising the small organicmolecule may be concentrated (e.g., by evaporation of at least a portionof the solvent)

The methods and/or compositions of the present invention may findapplication relating to pharmaceutical compositions and/or methods,wherein the molecular species is a pharmaceutically active agent. Aswill be known to those of ordinary skill in the art, differentpolymorphs of pharmaceutically active agents can have significantlydifferent properties including solubility, bioavailability, and/orstability Accordingly, the ability to control the formed polymorph ofthe pharmaceutically active agent (e.g., using the methods and systemsdescribed herein) provides the advantage of having the capability toform a selected polymorph based on the properties of the crystallizationsubstrate. For embodiments where the crystals of the pharmaceuticallyactive agent are not to be separated from the crystallization substrate(e.g., in embodiments wherein the pharmaceutically active agent iscrystallized in the pore of the substrate), the substrate may besubstantially non-toxic and/or bioabsorbable.

In some aspects of the present invention, methods are provided forforming a plurality of particles comprising a crystallized active agent(e.g., a crystallized pharmaceutically active agent). In some cases, thecrystallized active agent is formed in at least a plurality of thepores, e.g., according to the methods described herein. For example, insome cases, the method comprises providing a solution containing acrystallization substrate comprising plurality of polymeric particles,wherein the polymeric polymers are porous. The polymeric materials usedto form the polymeric particles may optionally be selected to comprise aplurality of functional groups to aid in the interaction between thepolymeric particles and the active agent. The polymeric particles may beexposed to a solution comprising the pharmaceutically active agent.Using the methods and systems described herein, the pharmaceuticallyactive agent may be crystallized in at least a portion of the pluralityof pores of the polymeric particles. For example, a solution may beprovided comprising the plurality of porous polymeric particles and thepharmaceutically active agent, and at least one method or system may beemployed to induce crystallization, as described above. A compositioncomprising a plurality of polymeric particles and a pharmaceuticallyactive agent crystallized in at least a portion of the pores may beformed and/or provided. The composition may be isolated and used in avariety of application. For example, for use in a pharmaceuticalcomposition for administration to a subject.

The compositions, methods, and systems of the present invention may befind advantageous use in applications involving crystallizedpharmaceutically active agents. Crystallization is a common techniqueused to purify pharmaceutically active agents in pharmaceuticalmanufacturing processes. Generally, after the pharmaceutically activeagent has been crystallized, the crystals are granulated and blendedwith excipients in a series of solid state operations. The granulationand blending steps may be problematic for example, as the steps may beplagued by poor process control ability and/or final product uniformity,and/or the process parameters may be very sensitive to the properties ofthe specific type of pharmaceutically active agent. In addition, bulkcrystallization of pharmaceutically active agents may not always providea single type of polymorph of the pharmaceutically active agent and/orchanges over time during a manufacturing process can cause differenttypes of polymorphs to form. For example, a slight change in temperaturemay cause a pharmaceutically active agent to crystallize in a phasedifferent than the desire phase. In addition, granulating and/orblending steps may induce changes in the crystal phase of thepharmaceutically active agent.

The methods, systems, and compositions as described may aid in reducingand/or eliminating typical processing steps such as granulation and/orblending, as well as reducing the probability that the pharmaceuticallyactive agent crystallizes in an undesired polymorphic form. For example,as described herein, the methods and systems of the present inventioncan be used to promote the crystallization of a small organic molecule(e.g., a pharmaceutically active agent) and a specific polymorph, thusreducing the likelihood of the formation of an undesired polymorph. Inaddition, a pharmaceutically active agent may be crystallized in aplurality of polymeric particles, and these particles may be useddirectly in a pharmaceutical composition (e.g., they may be bound toform a tablet), thus reducing/eliminate typical processing steps. Thus,the crystallization and/or nucleation techniques described herein canprovide a greater ability to control the crystal phase of thepharmaceutically active agent, and/or the reduce or eliminate processingsteps which may result in changes in the crystal phase that may occurduring these steps (e.g., see FIG. 2).

In some embodiments methods are provided for making a pharmaceuticalcomposition. In some cases, the method comprises crystallizing apharmaceutically active agent in the presence of at least one excipient(e.g., a crystallization substrate comprising a plurality of polymericparticles, optionally porous, and optionally comprising a plurality ofcomplimentary functional groups to the pharmaceutically active agent),forming a pharmaceutical composition comprising the pharmaceuticallyactive agent and the at least one excipient. In some embodiments, theprocess is free or essentially free of mechanical steps for altering thephysical properties of the pharmaceutically active agent or thepharmaceutical composition. In some cases, the process is essentiallyfree of mechanical steps for reducing particle size of thepharmaceutically active agent and at least one excipient. That is, thatthe pharmaceutical composition may be prepared without the need formechanical steps such as granulation and/or blending. The resultingpharmaceutical composition may be provided to a subject. In some cases,prior to administration to the subject, the pharmaceutical compositionmay be formed into a pharmaceutical product suitable for administration.For example, the particles may be contained in a capsule (e.g.,including gel capsules), as a tablet, in a solution (e.g., forinjection), etc.

In some embodiments, methods are provided for administering apharmaceutically active agent to a subject. In some cases, the methodcomprises providing a crystallization substrate comprising a pluralityof polymeric particles having a plurality of pores or features and apharmaceutically active agent crystallized in at least a portion of thepores or features; and administering the plurality of polymericparticles to the subject (e.g., a human). Methods and compositionscomprising a plurality of polymeric particles having a pharmaceuticallyactive agent crystallized in at least a portion of the pores or featuresare described herein.

The term “small molecule” is art-recognized and refers to a compositionwhich has a molecular weight of less than about 2000 g/mole, or lessthan about 1000 g/mole, and even less than about 500 g/mole. Smallmolecules may include, for example, nucleic acids, peptides,polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates,lipids or other organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention. The term “smallorganic molecule” refers to a small molecule that is often identified asbeing an organic or medicinal compound, and does not include moleculesthat are exclusively nucleic acids, peptides, or polypeptides. In somecases, the small organic molecule is a pharmaceutically active agent(i.e., a drug). A pharmaceutically active agent may be any bioactiveagent. In some embodiments, the pharmaceutically active agent may beselected from “Approved Drug Products with Therapeutic Equivalence andEvaluations,” published by the United States Food and DrugAdministration (F.D.A.) (the “Orange Book”). In a particular embodiment,the pharmaceutically active agent is aspirin or acetaminophen.

The compositions and/or crystals described herein may be used in“pharmaceutical compositions” or “pharmaceutically acceptable”compositions, which comprise a therapeutically effective amount of oneor more of the polymers or particles described herein, formulatedtogether with one or more pharmaceutically acceptable carriers,additives, and/or diluents. The pharmaceutical compositions describedherein may be useful for diagnosing, preventing, treating or managing adisease or bodily condition including conditions characterized byoxidative stress or otherwise benefitting from administration of anantioxidant. Non-limiting examples of diseases or conditionscharacterized by oxidative stress or otherwise benefitting fromadministration of an antioxidant include cancer, cardiovascular disease,diabetes, arthritis, wound healing, chronic inflammation, andneurodegenerative diseases such as Alzheimer Disease.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose structures, materials, compositions, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid, gel or solid filler, diluent, excipient, or solventencapsulating material, involved in carrying or transporting the subjectcompound, e.g., from a device or from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations.

As used herein, a “subject” or a “patient” refers to any mammal (e.g., ahuman), for example, a mammal that may be susceptible to a disease orbodily condition. Examples of subjects or patients include a human, anon-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cator a rodent such as a mouse, a rat, a hamster, or a guinea pig.Generally, the invention is directed toward use with humans. A subjectmay be a subject diagnosed with a certain disease or bodily condition orotherwise known to have a disease or bodily condition. In someembodiments, a subject may be diagnosed as, or known to be, at risk ofdeveloping a disease or bodily condition.

The following are herein incorporated by reference in their entirety forall purposes: U.S. Provisional Patent Application Ser. No. 61/375,925,filed Aug. 23, 2010, and entitled “Compositions, Methods, and SystemsRelating to Controlled Nucleation of Small Organic Molecules;” U.S.Provisional Patent Application Ser. No. 61/418,767, filed Dec. 1, 2010,and entitled “Compositions, Methods, and Systems Relating to ControlledNucleation of Small Organic Molecules;” and U.S. Provisional PatentApplication Ser. No. 61/466,759, filed Mar. 23, 2011, and entitled“Compositions, Methods, and Systems Relating to Controlled Nucleation ofSmall Organic Molecules.”

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

The following example provides both prophetic and working examples ofmethods and systems of the present invention. In this Example, exemplarymethods and systems for controlling the nucleation of small organiccompounds from solution by tuning the surface chemistry and morphologyof amorphous substrates are described. Polymers are synthesized withvarious surface chemistries and pore structures to expedite thenucleation of small organic molecules.

In many areas of science and technology, such as the production ofpharmaceuticals, semiconductors, and optics, as well as the formation ofbiominerals, the ability to control crystallization is desired.Nucleation is an important step in controlling the crystallizationprocess. Generally, crystallization starts with heterogeneous nucleationwhich occurs at random foreign surfaces.

Controlling Nucleation Kinetics and Nucleation Density by AdjustingSurface Chemistry:

Nucleation is an activated process. The presence of interfaces may alterthe free energy barrier of nucleation through various means (e.g., thedegree of interaction between the substrate and the crystallizingmolecule by adjusting the substrate surface chemistry).

To impart various surface chemistries, crosslinked polymers wereprepared to heterogeneously nucleate the active pharmaceuticalingredient (API, also herein described as a pharmaceutically activeagent) of interest. The polymers were crosslinked, resulting inminimized solvent uptake and stabilized surface functionality. Polymerswere synthesized via photo polymerization method. UV curable monomersthat could interact with the API of interest (e.g., via hydrogen bondsor pi-pi stacking) were selected.

One API employed was aspirin, and monomers with varying functionalgroups were selected and divided into a number of types according to themain functionality contained (e.g., see Scheme 1). Group (a) includes2-hydroxyethyl methacrylate (HEMA), acrylic acid (AA) and methacrylicacid (MAA) which contain carboxyl or hydroxyl groups that provide bothhydrogen bond donors and acceptors. Groups (b) and (c) include tertiaryamides and amines that are rich in hydrogen bond acceptors, and they areN,N,-dimethylacrylamide (DMAA), vinylpyrrolidone (VP),4-Acryloylmorpholine (AM) in group (b) and 4-vinylpyridine (4VP),vinylimidazole (VI), 2-dimethylamino ethyl methacrylate (DMAEMA) ingroup (c). Monomers in group (d), methyl methacylate (MMA) andtert-butyl methacrylate (t-BuMA), contain the carboxylic acid esterfunctionality that is also seen in aspirin. In group (e) are twophenyl-ring-containing monomers, styrene (STY) and chloromethylstyrene(CIMSTY).

To determine the relative nucleation activities of surfaces and toselect the surface chemistries which best promote nucleation, ascreening method was developed where the API of interest wascrystallized on the candidate polymer films by static isothermalcrystallization method and the nucleation area density on the polymerfilm was used as a parameter to indicate the nucleation activity ofsurfaces.

Specifically, the polymer films were synthesized by UV curing a mixtureof a monomer and a crosslinker, ethylene glycol dimethacrylate (EGDMA),then immersed in supersaturated aspirin-toluene solution to performstatic isothermal crystallization. The crystal densities on the polymerfilms are shown in FIG. 3.

In FIG. 3: Nucleation density of aspirin on polymer films. Columnsrepresenting polymers from groups (a), (b), (c), (d), (e) are numbered(i), (ii), (iii), (iv), and (v), respectively. Error bars were derivedfrom three repeats.

To further quantify the surface chemistry effect on nucleation kinetics,nucleation induction time distribution was measured with the API ofinterest. Nucleation induction time is an indicator of the surfacenucleation activity because it can be shortened when the presence of asurface lowers the free energy barrier of nucleation. Due to thestochastic nature of nucleation events, a significant number ofexperiments were performed to obtain the probability distribution ofnucleation induction time.

As an example of induction time measurements, polymer plates wereprepared by UV polymerizing the mixture of a monomer and the crosslinkerdivinylbenzene held by Teflon holders. Subsequent drying under vacuumremoved unreacted monomer molecules. Monomers used to synthesize polymerplates were 4-Acryloylmorpholine (AM), 4-Hydroxybutyl acrylate (HBA),2-Carboxyethyl acrylate (CEA), and styrene (STY). AM, HBA, and CEAserved as positive controls, whilst STY a negative control. Each polymerplate was inserted together with the Teflon holder vertically into theaspirin solution, and then the solution was quenched to initiatenucleation. The starting concentration and the nucleation temperaturewere chosen such that the supersaturation produced was high enough togive reasonably short induction time but low enough to suppress bulknucleation. 48 vials of each polymer sample were tested simultaneouslyand the fraction of vials crystallized was recorded as a function oftime to produce a plot of cumulative probability distribution ofinduction time, as shown in FIG. 4.

In FIG. 4: Cumulative probability distribution of nucleation inductiontime (FIG. 4A) and statistical analysis on the same data sets (FIG. 4B)obtained with polymers synthesized via bulk polymerization.Crystallization of aspirin was performed at supersaturation S=4.75. Thelinear regression in 2(b) follows the formula ln(P)=−t/τ to obtain theaverage induction time τ. P is the probability for no crystallizationevent to occur within time t. Since the STY, HBA and bulk samplesproduced comparable nucleation rate, only the STY data was regressed.The results of linear fitting are i) STY ln(P)=−t/243.9, R²=0.987; ii)CEA ln(P)=−t/113.6, R²=0.978; iii) HBA ln(P)=−t/38.2, R²=0.995.

Controlling Nucleation Kinetics by Adjusting Surface Morphology:

Besides surface chemistry, surface morphology, especially porousstructures, may also play an important role in controlling nucleationkinetics and polymorphism. However, no study has been reported oncontrolling nucleation kinetics of small organic compounds with polymersof nanoscopic pores, nor a rigorous experimental method to evaluate thenucleation activity of the porous material.

The methods used to make porous polymer substrates or microparticlesinclude photopolymerization induced phase separation (PIPS),polymerization with high molecular weight polymers or nanoemulsions asthe porogen, etc. The method to measure nucleation kinetics isaforementioned.

For example, by the PIPS method, the mixture of a monomer and thecrosslinker divinylbenzene dissolved in ethanol was subjected to UVirradiation to initiate the polymerization. As the molecular weight ofthe polymer increases to a critical point, dynamic asymmetry betweensolvent molecules and the polymer lead to nucleation and growth ofsolvent-rich regions within the polymer matrix. Subsequent evaporationof the solvent during the vacuum drying leaves pores on the polymerfilms. One common feature shared by most porous surfaces is a raisededge surrounding the pore region, which may be a sign of eruptionalbehavior caused by fast solvent evaporation under vacuum. As determinedby AFM, the pores found on polymer sample AM were around 100 nm in widthand 4 nm in depth. Comparatively, polymer sample HBA carried pores about50 nm in width and 5-8 nm in depth. The non-porous AM and HBA sampleswere synthesized by bulk polymerization method and used as a control andhad relatively smooth surfaces despite a few impurities. Table 1 showedthat the presence of specific pore structure on polymer sample AM andHBA greatly shortened the nucleation induction time, and hence expeditednucleation.

TABLE 1 Comparison of the average nucleation induction time of aspirinwith nonporous and porous polymers. Average induction time τ (h) Polymertype Superaturation AM HBA Non-porous S = 4.75 38.2 243.9 Porous S =4.2  8.8 54.6

Controlling Crystal Orientation Via Specific Molecular Interactions:

From the manufacturing point of view, it is desired to control thecrystal morphology by designing the nucleation substrate. Researchershave oriented the Calcite crystals via electrostatic interactions andepitaxial relationships in the aqueous system. However, no study appearsto have been completed on the orientation of small organic molecules onamorphous polymeric substrates. In this example, the API of interest washeterogeneously crystallized on polymer surfaces of various chemistries.The preferred orientation of the API crystal with respect to the topsurface of the polymer film was identified with XRD and compared withbulk crystals. The configuration in the XRD measurement was such thatonly the crystal planes parallel to the polymer film surface was seen bythe X-ray, thus the peak significantly more intense relative to thereference corresponds to the preferred nucleation face.

In a specific instance, the orientation of aspirin crystals on polymerfilms AM, CEA, and HBA was investigated. The representative XRD patternsare shown in FIG. 5. It is apparent from FIG. 5 that aspirin (011) facepreferentially grows from polymer films HBA and AM, while (100) face onCEA. This result may be attributed, at least in part, to the underlyingmolecular interactions that steers the crystal orientation. Themolecular structure of the corresponding crystal facet is shown in FIG.18. The presence of carbonyl groups on aspirin (100) plane providespredominant hydrogen bond acceptors, and thus imparts slight Lewisbasicity to the facet. Comparatively, the (011) plane is more acidicwith the presence of undimerised carboxylic acid functional groups.Since CEA polymer surface mainly bares carboxyl groups, it is sensiblefor it to nucleate the (100) facet via hydrogen bonds with carbonylgroups. As for AM and HBA polymer surfaces which are rich inhydrogen-bond acceptors, they could preferentially nucleate the (011)facet through hydrogen bonds with carboxyl groups.

In FIG. 5: X-ray diffraction patterns of aspirin crystals nucleated frompolymer surfaces and from the bulk. The (hkl) indices of thecrystallographic planes are labeled over corresponding peaks. The broadpeak around 20° seen in the top three diffraction patterns is attributedto the amorphous polymer film. Two primary peaks were observed in allthe four patterns, one around 7.7°, and the other around 15.6°. Sincethe (011) peak is separated from the (002) peak by a 2θ angle of only0.17 degree, the 2θ angle differences between the two primary peaks werecarefully measured to determine that the peak around 15.6° matched with(011) plane. Current industrial practice to control nucleation fromsolution involves adjusting the nucleation temperature profile,supersaturation level, solvent to crystallize from, impeller designs,stirring speed, seeding, etc. However, the nucleation behavior remainslargely unpredictable due to the presence of unregulated foreignsurfaces present on impurities, the vessel wall or the impeller, becausethe foreign surfaces may possess the surface properties that happen tolower the nucleation energy barrier, etc. The methods described hereinallows for the utilization of foreign surfaces with the surfacechemistry and morphology designed to regulate the nucleation kineticsand crystal outcome.

Crystalline materials have been extensively studied for controllingcrystallization on foreign surfaces. The epitaxy mechanism is welldeveloped for nucleation on crystalline surfaces, such as self-assembledmonolayers or SAMs, molecular single-crystal surfaces, crystallinepolymer surfaces, etc. Compared with crystalline surfaces, amorphoussurfaces such as crosslinked polymers as demonstrated in herein areeasier, cheaper to fabricate, and the manufacturing protocol is alreadywell established in the industrial practice. Furthermore, there is muchgreater flexibility to achieve complex morphologies with various surfacechemistry, and there is no system compatibility issue with thecrosslinked polymers, allowing the usage of virtually any solvent,unlike in the case of molecular crystals as the substrates.

This section focuses on the role of surfaces in crystallization from theperspective of nucleation kinetics, nucleation density, and crystalorientation in a quantitative manner, which is not reported before forsmall organic molecules. A method to systemically evaluate thenucleation activity of surfaces with various chemistry and morphology isdemonstrated, which is a valuable tool to aid the design and selectionof nucleation active surfaces.

Applications:

The method described herein may be applied to designing interfaces orparticles to regulate nucleation kinetics, to control the nucleationdensity and crystal orientation for pharmaceutical industry, foodindustry and other industries that require crystallization of smallorganic compounds.

1. Application in pharmaceutical manufacturing: Crystallization isextensively used to purify the active pharmaceutical ingredients in thepharmaceutical manufacturing process. After the crystallization step,the API crystals are generally granulated and blended with excipients ina series of solid state operations. The granulation and blending stepscan be problematic. For example, they may be plagued by poor processcontrollability and final product uniformity, the process parameters maybe sensitive to the properties of the drug crystals, etc. On the otherhand, the properties of the drug crystals are constantly varying due tothe difficulty in controlling crystallization.

The methods described herein allow for heterogeneous crystallization ofAPI from solution on the surface of an amorphous excipient, so that thesubsequent API compaction, granulation and blending with excipients canbe ultimately eliminated or reduced. Furthermore, API nucleationkinetics and final crystal form have the potential to be tuned bydesigning the excipient surface properties.

2. Application in drug delivery: Recent years have seen great enthusiasmin making nanoscopic drug particles to improve the bioavailability ofhydrophobic compounds and to release the drug in a controlled mannerfrom a biocompatible nanoporous matrix. Generally, the drug particlesare either broken down mechanically to reduce to desired sizes or arephysically absorbed into nanoporous matrix in amorphous state. However,these methods are not ideal because the drugs are prone to phasetransformation under mechanical stress or to recrystallize since theamorphous form is metastable. The methods described here allow fordirect crystallization in the drug carrier and of making nanocrystals innanoporous polymer matrix to enhance drug availability.

Example 2

The following example provides both prophetic and working examples ofmethods and systems of the present invention. This example describesexemplary methods and systems for the nanostructure and chemical makeupof polymer particles to control nucleation from solution. The methodsand system may allow for the synthesis of unique composite particlescomprised of crystalline active pharmaceutical ingredient and polymericexcipients.

As described above, in many areas of science and technology, such as theproduction of pharmaceuticals, semiconductors and optics, as well as theformation of biominerals, the ability to control crystallization isdesired

Synthesis of Porous Polymer Particles with Controlled PorousMicrostructure:

The polymer particles involved in this example are hydrogels. Hydrogelswill be known to those of ordinary kill in the art and are generallydefined by a chemically cross-linked network which swells in thepresence of solvent such that the total volume fraction of polymer inthe microstructure is much less than unity (e.g., typically less than50%). Because mass transport and adsorption of chemical species withinpolymer hydrogels is important to their material function, in someembodiments, the porosity (pore structure) of hydrogels may be acritical aspect to their applications. Typically, porosity iscategorized based on the length scale of the pore structure thatcontrols various properties of the material. In this example, the termmicropores refers to pores having characteristic dimensions less than 10nanometers (e.g., 1-10 nm), which may influence the rate of diffusion ofmolecular species within the hydrogel interior. Similarly, the termmesopores refers to pores having dimensions from 10-1000 nanometers, andmay primarily influence the overall surface area/volume ratio of thematerial, and control the diffusion of colloidal species within themicrostructure. The term macropores refers to pores having dimensionsfrom 1-100 microns may, which also may influence surface area/volumeratio, and may allow for flow and convective mass transport through themicrostructure.

The polymer particles used in this example are chemically cross-linkedhydrogels of poly(ethylene glycol) diacrylate with cube-like shape anddimensions of 30 μm×30 μm×23 μm prepared by Stop-Flow Lithography (SFL)(e.g., see FIG. 6A). The polymerizing fluid used was a composition ofpoly(ethylene glycol) diacrylate (PEG_(n)DA) as the monomer at variousconcentrations, 25 vol % PEG_(n) as a molecular porogen (see below), and5 vol % Darocur 1173 photoinitiator, with the remainder being ethanol.Upon irradiation with patterned UV light during SFL, the fluid undergoesfree radical polymerization to produce a lithographically patternedcross-linked hydrogel microstructure.

A primary aspect of this example involves development of a method bywhich these various levels of porosity may be independently controlled,and thus allowing for the synthesis of polymer hydrogel particles withwidely tunable porous structure. Although methods currently exist tocontrol one of these levels of porosity, no methods currently exist tocontrol all three levels of porosity independently and at the same time.Previous methods use photolithography of a polymerizable fluid within amicrofluidic device to create polymer microparticles and microstructuresof well-defined shape (e.g., see FIG. 6). For example, FIG. 6A showscube-like hydrogel microparticles created by SFL (see details below). Inthis example, SFL is also used to create lithographically patternedmacroporous features within the particles. For example, FIG. 6B showstablet-shaped microparticles of the same composition as those in FIG.6A, but with regular arrays of square or circle-shaped holes ofdifferent size (e.g., down to about 2 microns) and density (e.g., up to60% of the projected area of the particle).

In FIG. 6: Optical micrographs of hydrogel particles produced by theinvented method: (FIG. 6A) cube-like particles with controlledmicroporous structure; (FIG. 6B) tablet-shaped particles with regulararrays of macroporous features and emulsion-templated mesopores; (FIG.6C) tablet-shaped particles with emulsion-templated macropores with anaverage pore size of 0.8 micron (determined by image analysis).

Mesoporous structure may be controlled by addition of an inert,non-polymerizable templating agent to the polymerizing fluid, which canbe removed upon completion of the polymerization to yield voids withsimilar size and shape of the templating agent. Emulsion droplets arefrequently used as templating agents, as they are inexpensive, simple toprepare, and can be used to create large volume fractions of voidswithin the polymer microstructure. However, previous methods forincorporating emulsion droplets into a polymerizable fluid suffer twodrawbacks. First, contemporary methods used to prepare the emulsion(e.g. mechanical mixing and ultrasonication) typically produce dropletsin the range of 1-100 microns, which are inappropriate for templating ofmesopores, and in the current method would interfere with macroporeformation. Second, these methods typically produce droplets withsignificant polydispersity (CV>100%), which prevents the ability toprecisely control the templated pore size.

In the current method, high-pressure homogenization is used to produce“nanoemulsion” droplets characterized by low polydispersity and sizeappropriate for mesopores (e.g., less than 0.5 micron). FIG. 7 showsthat high-pressure homogenization can be used to prepare nanoemulsionsof inert silicone oil droplets suspended in a polymerizable fluid (e.g.,as described herein) with controlled average drop size (in the range of100-600 nm) and low polydispersity (CV<0.3). To demonstrate the use ofemulsion-impregnated polymerizable fluids in the current method, FIG. 6Bshows macroporous tablet-shaped particles polymerized in the presence ofnanoemulsion droplets with an average diameter of about 110 nm at avolume fraction of 33%, demonstrating that the presence of the emulsiondroplets does not significantly affect the ability to form macroporousfeatures through lithographic patterning. Because the induced mesoporesin FIG. 6B are not visible by bright field optical microscopy, FIG. 6Cshows particles in which the emulsion droplets used to template poreshave average size of about 570 nm and volume fraction of 33%, showingthat the nanoemulsions can impart uniform pores within the particleinterior.

In FIG. 7: Average size of nanoemulsions droplets (determined by dynamiclight scattering) comprised of silicone oil with sodium dodecyl sulfatestabilizer produced by high-pressure homogenization versus appliedhomogenization pressure. Error bars represent coefficient of variationof the measured drop size distribution.

The porosity of cross-linked polymer hydrogels is typicallycharacterized by the mesh size, which is related to the average polymermolecular weight between chemical cross-links. In the current method,this quantity may be controlled by adjusting the composition of thepolymerizing fluid, including changes in the concentration and molecularweight of the monomer (e.g., in this case, PEG_(n)DA) as well asaddition of porogens (e.g., in this case, PEG_(n)) which can act asmolecular pore-templating agents. To demonstrate this ability, FIG. 8shows the estimated mesh size of hydrogel particles obtained fromequilibrium swelling measurements for a series of PEGDA molecular weightand concentration polymerized in the presence of PEG_(n) with M_(n) of200 g/mol, yielding control of the mesh size in the range of 0.5-2.5 nm.Furthermore, when combined with other levels of porosity, the describedmethod can produce polymer hydrogel particles with combined porosity ofup to 95%, providing a large specific surface area while maintainingmechanical integrity of the particle.

In FIG. 7: Mesh size of PEGDA hydrogel particles suspended in 38/62ethanol/water (v/v) measured by equilibrium swelling measurements as afunction of PEGDA concentration (% v/v) and molecular weight of the PEGchain (M_(n)) in the pre-polymer solution.

Controlling the Nucleation Kinetics by Adjusting the Polymer MicroporousStructure:

Surface morphology, especially porous structure, can play an importantrole in controlling nucleation kinetics and polymorphism in someembodiments. However, no study has been reported on controlling kineticsof nucleation from solution with polymers of tunable microstructure.Recent studies have shown mesoporous silica with 5-10 nm pores inducedprotein crystallization from aqueous solution. On the other hand,crystallization of benzyl alcohol and o-terphenyl from melt wassuppressed in controlled pore glasses with 8.5 nm and 4 nm pores. AMonto Carlo simulation of nucleation in a square shaped open pore with2D Ising model for one component system indicated the existence of anoptimum pore size corresponding to a maximal nucleation rate. However,it has not been experimentally studied how the rate of nucleation fromsolution depends on the pore sizes. Moreover, the effect of porechemistry on nucleation was largely neglected. Overall, mechanisticunderstanding is inadequate on nucleation from solution innanoconfinement, which is a necessity for designing polymers with theproper microstructure and chemistry to control crystallization.

The effect of nanoconfinement and pore chemistry on nucleation kineticswas investigated using a series of cube-like PEGDA hydrogelmicroparticles with M_(n)=130, 200, 400, 575, and 700 synthesized by themethod described herein. The hydrogels are denoted with theircorresponding M_(n) of the monomers they are synthesized from for therest of this example. In the crystallization solvent, the mesh sizes ofthese hydrogels range from 0.8 nm to 2.0 nm, as listed in Table 2.Aspirin (ASA) and acetaminophen (ACM) were used as model compounds forthe crystallization studies. Crystallization of ASA or ACM from a 62/38v/v water-ethanol mixture was induced by cooling, with or without PEGDAparticles suspended in the solution by stirring. FIG. 9 shows an opticalmicroscopy image of ASA crystals on PEG₇₀₀DA particles as crystallizedfrom 38 mg/ml ASA solution in 38/62 ethanol/water (v/v) with 15 μg/mLPEG₇₀₀DA particles at 15° C., solution stirred at 700 rpm.

Nucleation kinetics of model compounds was investigated by measuring thenucleation induction time probability distribution. Nucleation inductiontime is generally time elapsed prior to the formation of a detectableamount of the new crystalline phase. It is a useful indicator of thesurface nucleation activity because it can be dramatically shortenedwhen the presence of an interface lowers the free energy barrier ofnucleation. Due to the stochastic nature of nucleation events, a largenumber of experiments were performed to obtain the probabilitydistribution of nucleation induction time. To obtain the averageinduction time τ, statistical analysis on the induction time data wasconducted based on the understanding that nucleation follows a Poissondistribution. According to Poisson statistics, the probability for anucleation event to occur beyond time t is P=exp(−t/τ), which impliesthat the fraction of vials without nucleation at time t exponentiallydecays as a function of time, where the scaling factor for time is theaverage induction time.

The statistical analysis of ASA nucleation induction time with orwithout PEGDA particles with a series of mesh sizes (denoted by M_(n))suspended in a supersaturated aspirin solution was determined. Thecalculated average induction times are summarized in Table 2. Almost allPEGDA particles successfully promoted aspirin nucleation, except forM_(n)=130 g/mol. Specifically, the addition of particles with M_(n)=400g/mol to the aspirin solution dramatically reduced the aspirinnucleation induction time to 60 minutes, while at the same experimentalconditions, no nucleation event was detected in the absence of particle400. Furthermore, the nucleation activity of the PEGDA particlesdecreased sharply by either increasing or decreasing the mesh size asindicated by the average induction times summarized in Table 2. Thisobservation suggests that there exists an optimum mesh size forexpediting nucleation from solution.

For determining the statistical analysis of aspirin nucleation inductiontime with PEGDA particles of various mesh sizes (particles denoted byM_(n)), 1.9 mL of 38 mg/ml ASA solution in 38/62 ethanol/water (v/v)with 15 μg/mL PEGDA particles was cooled at a rate of 5° C./min from 35°C. to 15° C. to achieve a supersaturation of 2.1. The solution wasstirred at 700 rpm. The onset of nucleation was detected by an IR probewhich measures the transmission signal through the solution. A total of10 vials were cycled for 5-10 times to yield 50-100 nucleation inductiontime measurements for the statistical analysis.

Comparison of ASA nucleation rates in the presence of PEGDA particles ofvarious mesh sizes at supersaturation of 2.1 and 3.3 were determined.The nucleation rate, J, is given by J=1/τV, where τ the averageinduction time (Table 1) and V the volume of solution. For this equationto be valid, the assumption is that the crystal growth rate is muchfaster than the nucleation rate, which is the case for ASA. At asupersaturation of 2.1, the crystallization temperature was 15° C.,solution volume 1.9 mL; for a supersaturation of 3.3, thecrystallization temperature was 8° C., solution volume 1.0 mL. Othercrystallization conditions were kept the same.

Controlling nucleation kinetics by tuning the API-polymer interaction:Without wishing to be bound by theory, the success of the PEGDAparticles in facilitating ASA nucleation may result from favorableinteractions between aspirin and the PEGDA polymer matrix in thesolution environment. This favorable interaction may have a positiveimpact on API nucleation in two ways. Firstly, may it lead topreferential partitioning of API into the PEGDA hydrogel interiorrelative to the bulk solution; hence, in the PEGDA phase, a higher APIsupersaturation may be achieved that favors nucleation. Secondly, it mayreduce the free energy barrier associated with creating new interfacesduring nucleus formation by replacing the nucleus-solvent interface withthe nucleus-polymer interface, which may lower the free energy due tofavorable interactions between the nucleus and the polymer.

This theory is exemplified by quantifying the partitioning of aspirinbetween the PEGDA gel phase and 38/62 ethanol/water (v/v), anddetermining the concentration of aspirin in the hydrogel particles. Thepartitioning experiments were performed at the same solutionconcentration and temperature as in the nucleation induction timeexperiments. In the partitioning experiment, both the concentrations ofaspirin and ethanol were measured, as there are three species in thebulk solution, namely aspirin, ethanol and water, all of which partitionin the gel phase to a certain degree determined by their relativeinteraction with the PEGDA polymer matrix. All the PEGDA particlesconcentrated aspirin for more than three times with respect to the bulk,while the ethanol concentrations remained comparable to that of thebulk. A slight increase in the aspirin concentration was observed asM_(n) of the PEGDA particles increases from 130 g/mol to 700 g/mol. Thisphenomenon may be due, at least in part, to the fact that thecrosslinking points and the subchain in the polymer matrix possessdifferent chemical functionalities, hence they differ slightly in theirdegrees of interaction with aspirin and solvent molecules. Overall, thepartitioning is consistently high for all the PEGDA particles andremained relatively insensitive to the variation in polymer mesh size.This result indicates that the interaction between aspirin molecules andthe polymer matrix is favorable as compared to that between aspirin andsolvent.

This theory was further exemplified in that the effectiveness of PEGDAparticles in promoting API nucleation from solution was correlated withthe interaction between the PEGDA and API. A second API compound whichhas weaker interactions with PEGDA was tested and the nucleationactivity of the particles in this new system were assessed by performingnucleation induction time measurements. The study on a new API systemalso helps to demonstrate the generality of the existence of an optimummesh size corresponding to the highest nucleation rate. The chosen APIis acetaminophen (ACM), which was less concentrated in the PEGDA gelphase as compared to aspirin (FIG. 10), indicating a weaker interactionwith the PEGDA polymer matrix.

Nucleation induction time measurements on the acetaminophen system)further attest to the overall success of PEGDA particles in facilitatingnucleation. Table 1 shows that in most cases, the addition of particlesin the acetaminophen solution led to a shorter average induction timecompared with the bulk. This is not surprising given that acetaminophenpartitions in PEGDA particles to approximately twice the bulkconcentration (FIG. 10), thereby increasing the supersaturation leadingto more favorable crystallization conditions for acetaminophen.

Furthermore, as in the aspirin system, an optimum pore size was alsoobserved corresponding to the shortest nucleation induction time.However, unlike the aspirin system, the effect of PEGDA particles wasnot affected as dramatic in the case of acetaminophen as evidenced bythe following observations. Firstly, the addition of PEGDA particles atbest resulted in about ten-fold enhancement in the nucleation kineticsof acetaminophen, while in the case of aspirin, the degree ofenhancement is by many orders of magnitudes (Table 2). Secondly, theparticles were unable to induce acetaminophen nucleation at asupersaturation less than 3.7 within the experimental time frame, and atthis supersaturation level, bulk nucleation started to occur at adetectable frequency, implying that the solution was fairly close to theupper bound of the metastable zone. As for aspirin, the PEGDA particlesbegan to show effects at a much lower supersaturation (2.1). The factthat there was no detectable bulk nucleation at these conditionsindicates that the solution was far from the boundary of the metastablezone, in these embodiments. These observations suggest that the PEGDAparticles are less effective in inducing acetaminophen nucleation due todecreased partitioning compared with aspirin.

The results discussed above supported the theory that in addition totheir nanostructures, the nucleation activity of the crosslinkedpolymeric particles also their interactions with the API. The mechanismof PEGDA particle-induced nucleation was partially explained by thehigher API supersaturation inside the particles due to the effect ofpreferential partitioning of the API.

Composition of aspirin solution in the PEGDA gel phase were comparedwith the bulk phase. PEGDA gels sufficiently large for convenienthandling were synthesized by UV polymerization, following the sameformulation as the synthesis of PEGDA particles used in thecrystallization study. The PEGDA gels were washed with ethanol andvacuum dried to remove unreacted species. The gel was then immersed inexcessive 38 mg/ml aspirin solution in 38/62 ethanol/water (v/v) andallowed sufficient time to reach equilibrium swelling at 15° C. beforetaken out, pad dried, and put in excessive water. After 5 hours immersedin water, the concentrations of aspirin and ethanol in the aqueous phasewere analyzed by UV-Vis spectroscopy and Gas Chromatography,respectively.

In FIG. 10: Partitioning of aspirin vs. acetaminophen in the PEGDA gel.Y axis represents the relative API concentration in the PEGDA,normalized by the bulk concentration. The aspirin partitioningexperiment was conducted in 38 mg/ml aspirin solution in 38/62ethanol/water (v/v) at 15° C. As for acetaminophen, 95 mg/mlacetaminophen solution in 38/62 ethanol/water (v/v) was used and theequilibration temperature was 8° C. In both cases, the experimentalconditions were kept the same as used in the nucleation induction timestudy.

Statistical analysis of acetaminophen nucleation induction time withPEGDA particles was determined as follows: 1 ml 95 mg/ml aspirinsolution in 38/62 ethanol/water (v/v) with 15 ug/ml PEGDA particles wascooled at a rate of 5° C./min from 35° C. to 8° C. to achieve asupersaturation of 3.7. The solution was stirred at 700 rpm. The onsetof nucleation was detected by an IR probe which measures thetransmission signal through the solution. A total of 10 vials werecycled for 5-10 times to yield 50-100 nucleation induction time data forthe statistical analysis.

TABLE 2 Comparison of the average nucleation induction times (τ) for APIcrystallization with PEGDA microparticles of various mesh sizes. Theaspirin (ASA) crystallization was performed at two supersaturationlevels (S), 2.1 and 3.3. The acetaminophen (ACM) crystallization wasconducted at the supersaturation of 3.7. The PEGDA particle mesh sizeswere calculated from the swelling ratio in 38/62 ethanol/water (v/v)based on the Flory-Rehner theory for swollen cross-linked gels. Thestandard errors of average induction times were calculated from thestandard error values for the slopes regressed from the lnP vs. t plots(FIG. 6) following the formula lnP = −t/τ. PEGDA Bulk M_(n)(g/mol) (noparticles) 130 200 400 575 700 Mesh size N/A  0.8 ± 0.1  1.0 ± 0.1 1.5 ±0.1 1.8 ± 0.1 2.0 ± 0.1 (nm) ASA τ (min) ∞ ∞ 910 ± 40 63 ± 3  1900 ±100  6600 ± 1100 S = 2.1 ASA τ (min) 2500 ± 600  330 ± 60 52 ± 3 123 ±7  NA 240 ± 20  S = 3.3 ACM τ (min) 6500 ± 1600 920 ± 70 540 ± 20 1720 ±150  NA 6500 ± 1600 S = 3.7

For the comparison of nucleation rates of aspirin and acetaminophen:Nucleation rate is in the number of nucleus per unit time per unitvolume, as calculated from the average induction time by J=1/τV, where Jis the nucleation rate, τ the average induction time, V the volume ofsolution. For this equation to be valid, the assumption is that thecrystal growth rate is much faster than the nucleation rate, which istrue for both aspirin and acetaminophen. The nucleation rate correspondsto supersaturation of 2.1 for aspirin, and 3.7 for acetaminophen.

Design Principles for Making API-Excipient Composite Particles:

Based on the fundamental understanding obtained from the above describedmethods and systems, general guidelines may be followed and design thechemistry and the structure of excipient and for selecting anappropriate solvent in order to maximize the likelihood forcrystallizing a given API on/in the excipient.

To facilitate API nucleation on the excipient, the chemical makeup ofthe excipient and the solvent should be selected such that the APIinteracts stronger with the excipient than with solvent molecules (theinteraction criterion). In the cases studied here, this was achieved byinteractions between the polymer and API that led to equilibriumpartitioning of API into the hydrogel particle phase relative to bulksolution. This yields increased supersaturation within the hydrogelinterior, which is a significant driving force in classical theories ofnucleation. Further tuning of the polymer chemistry to enhance thiseffect for a given API may be achieved by incorporating other chemicalfunctional groups into the polymer network by co-polymerization ofPEG_(n)DA with other species capable of participating in thephotopolymerization used to create the particles.

Shown in FIG. 11: Hansen parameters can aid the selection of the polymerchemistry that satisfy the interaction criterion. The distance ‘d’ inthe Hansen parameter space provides a semi-quantitative measure for theextent of interaction. ‘d’ has been shown to be correlated with ‘κ’, thepartition coefficient of API in the polymer gel from solution, whichserves to measure the interaction between API and the polymer. Theinteraction between the API and solvent is indicated from the APIsolubility, and that between the polymer and the solvent from gelswelling. According to the ‘Interaction Criterion’, it is desired tohave API partitioning as high as possible, and API solubility and gelswelling as low as possible. However, practically speaking, a stablemicrogel suspension is needed as well as a reasonable API crystal yield,which require high degree of gel swelling and high API solubility. Tobalance the two requirements, the second criterion for selectingappropriate polymer chemistry is to have intermediate gel swelling andAPI solubility.

Furthermore, the rate of crystallization was found to vary by orders ofmagnitude depending on the mesh size of the cross-linked polymer networkwithin the particle. This suggests that the particle microstructure canbe rationally tuned for a specific API in embodiments where the criticalnucleus size is known, and can act as a screening tool for favorablenucleation conditions if it is not known. Additionally, the mesh sizecan be controlled by a number of factors in the particle synthesismethod, including the concentration and molecular weight of both monomerand molecular porogen species, as well as the conversion of thepolymerization reaction. Thus, the mesh size can be controlled over avery wide range while at the same time providing flexibility to changesin other properties of the excipient particles.

Current industrial practice to control nucleation from solution involvesadjusting the nucleation temperature profile, supersaturation level,crystallization solvent, impeller designs, stirring speed, seeding withexisting API crystals, etc. However, the nucleation behavior remainslargely unpredictable due to the presence of unregulated foreignsurfaces present on impurities, the vessel wall or the impeller, becausethe foreign surfaces may possess characteristics that happen to lowerthe nucleation energy barrier. The methods and systems described hereinenable utilization of excipient particles with surface chemistry andmorphology designed specifically to directly control the nucleationkinetics and crystal outcome.

Crosslinked polymers and functionalized glass have been used to controlthe polymorphs of pharmaceutically related small molecular compound.However, the methods described herein use the role of surfaces incrystallization from the perspective of nucleation kinetics, nucleationdensity and crystal orientation in a quantitative manner, which is notreported before for small organic molecules. Furthermore, methods tosystemically evaluate the nucleation activity of surfaces with variouschemistry and morphology were provided, which is a valuable tool to aidthe design and selection of nucleation active surfaces.

Crystalline materials have been extensively studied for controllingcrystallization on foreign surfaces. The epitaxy mechanism is welldeveloped for nucleation on crystalline surfaces, such as self-assembledmonolayers (SAMs), molecular single-crystal surfaces, crystallinepolymer surfaces, etc. A primary disadvantage of crystallization onthese materials is that the resulting crystals are immobilized to asurface, and generally are broken if they are to be used in furtherprocess steps. This effects the crystal size distribution, which may bedetrimental to the eventual formulation of the substance. Because themethods/systems described herein use suspendable particles as theexcipient surface, the particles can easily be flowed through devicesand further process components while maintaining the mechanicalintegrity of the particle and API crystals.

Furthermore, compared with crystalline surfaces, amorphous surfaces suchas the cross-linker polymers demonstrated here are easier and cheaper tofabricate. The synthesis method described herein for polymer excipientsis also much more flexible toward excipients with complex morphologiesand different surface chemistries. Because the polymers particles arecross-linked, they can be used with virtually any solvent, unlike thecase of molecular crystal excipients, for which the solvent should becarefully chosen so that neither excipient nor API is soluble under thecrystallization conditions.

Finally, the ability of the polymer hydrogel excipients to absorb andconcentrate API due to equilibrium partitioning within the interior ofthe particles is a novel and unique ability. In other methods, theproduction of a commensurate increase in supersaturation is either byadding more API to solution, which may not be possible if the substanceis scarce, or by changing the solvent in or temperature at which thecrystallization is performed, which may result in incompatibilities withsubsequent process steps.

In this example, the optimum mesh size for inducing ASA nucleation wasfound to be approximately 15 Å, and the diameter of ASA molecules about6 Å (estimated from the crystal density). Without wishing to be bound bytheory, the optimum mesh size may allow for aspirin molecules associatedwith polymer chains to come within sufficient proximity to form anucleus, given the proper orientation (as would also be the case withACM). However, as the mesh size becomes smaller, a solute ‘sees’ morepolymer chains than other solute molecules, which may prevent theformation of large enough solute clusters; for larger mesh sizes, thesolutes associated with the polymer chain are further separated fromeach other, hence the solute-solute interaction may not be enhanced.Therefore, in some embodiment, the ability to control nucleation bynanoconfinement may lie in manipulating the effective solute-soluteinteraction, which can be strongly affected by polymer-soluteinteractions and the spatial confinement imposed by the polymermicrostructure, the interplay of which can give rise to the observedoptimum mesh size for expediting nucleation.

Accordingly, a series of experiments were performed in which the ASAcrystallization temperature was lowered from 15° C. to 8° C., therebyincreasing the supersaturation from 2.1 to 3.4. Since this change insupersaturation is significant whilst the absolute temperature was onlyaltered by 2%, this experiment primarily probes the effect of increasedsupersaturation, which may enhance effective solute-solute interactionsdue to increased density fluctuations. As a result, the observed optimummesh size decreased from 15 Å to 10 Å at the higher supersaturationlevel (Table 2). Accordingly, in this experiment, fewer solute moleculeswere needed to overcome the nucleation barrier, which was lowered due tohigher density fluctuations.

Example 3

It is well recognized that surfaces may play an important role inliquid-solid phase transformations, and surface morphology has beenshown to impact nucleation and crystallization. However, currentfundamental understanding is insufficient to allow the rational designof surfaces for nucleation/crystallization control. It is generallyaccepted that surface roughness helps promote nucleation, althoughlittle is known of the role that cavity shape of the rough surface playsin the nucleation process. This example shows that the shape of surfacenanopores (e.g., which are described in the specification as features orwells) can affect the nucleation behavior. Contrary to common belief, arough surface may inhibit nucleation of a molecular crystal fromsolution depending on surface morphology. The role played by surfacechemistry in nanopore-induced nucleation, in some embodiments, isdemonstrated in this example. Direction regarding surface-inducedcrystallization is provided, which may be applied to many areas ofscience and technology from designing ‘seed’ particles for regulatingcrystallization of various fine chemicals, to controlling pharmaceuticalpolymorphism, to orient biominerals on organic substrates, to promoteprotein nucleation for structure determination, and/or to inhibit icenucleation on airplanes.

Crystallization from solution generally initiates from a solid-liquidinterface, and “bulk” nucleation is generally thought to occur onmicroscopic surface in the liquid phase, However, various surfaceproperties impact nucleation have not been well understood, particularlyat the microscopic level. It is widely accepted and presented in theliterature that roughening of the surface present in the crystallizationsystem leads to accelerated nucleation, and in industrial practice,surface scratching has long been used to promote nucleation. However,without knowledge of the topological features of the surface cavities ata microscopic scale relevant to nucleation, the surface roughness alone,as a macroscopic parameter, may be insufficient, and even misleading, todescribe the effect of surface morphology on nucleation. Recently, therehas been an increase in the number of studies on crystal nucleation insub-100 nm pores, which were demonstrated to affect nucleation kinetics,polymorphism, and/or crystal orientation. These studies focused mainlyon the effect of pore size in the context of nanoscopic confinement, butthe role of pore shape was not studied.

This example demonstrates that nanopore shape can play a key role indetermining the kinetics of nucleation from solution. The importance offavorable surface chemistry, in some embodiments, in mediating theobserved ‘pore shape effect’ is also demonstrated.

In this example, the effects of angular pores are compared to those ofspherical pores of similar size. For this purpose, a fabricationtechnique was required to control both the feature geometry and the poresize down to length scales relevant to nucleation, i.e., to enablesurface patterning with pores from a few to hundreds of nanometers.Nanoscopic pores with high area density may be preferred, providing asufficient number of pores to ensure statistical significance of theobserved effects on nucleation. Sub-10 nm pores were avoided in thisexample because, in some cases, reported volume confinement effects onnucleation may mask the effects of pore shape. In addition, theresolution requirement for the fabrication technique was set by thelength scale of molecular events preceding nucleation, namely themolecular clustering and re-orientation that occur in domains of,probably, a few nanometers for small organic molecules. To meet theserequirements, a Nanoparticle Imprint Lithography′ (NpIL) technology wasdeveloped, which was used to fabricate nanopatterned polymer surfaceswith nanopore arrays of various shapes ranging from ten to hundreds ofnanometers, using nanoparticle assemblies as templates.

The fabrication of polymer films with spherical nanopores by NpIL isillustrated in FIG. 12. First, spherical silica nanoparticles wereself-assembled on a quartz slide driven by capillary forces during waterevaporation, and then anchored to the substrate via calcination to formthe imprint mold (FIG. 12A). Second, a mixture of monomer, crosslinkerand initiator was sandwiched between the imprint mold and the substrate,and subsequently polymerized under UV irradiation. The imprint mold wasthen easily peeled off to reveal a polymer film conforming to thesubstrate, with the nanopattern inversely transferred from the imprintmold (FIG. 12B). Polymer films with spherical nanopores ranging from 15nm to 300 nm were fabricated in this manner (FIG. 12C), templated bycommercially available monodispersed colloidal silica of various sizes.This method combines many of the advantages of NSL andultraviolet-assisted NIL, such as low cost, high throughput, andhigh-resolution. Moreover, in contrast to the commonly practiced NSLtechnique, where hydrofluoric acid is needed to dissolve the silicananoparticles, the above-described method removes the templatenondestructively by a simple liftoff from the polymer film, allowing themask to be recovered easily and reused.

In FIG. 12: Fabrication of polymer films with spherical nanopores byNpIL; (FIG. 12A) Template preparation via colloidal silica self-assemblyand its anchoring to the quartz substrate; (FIG. 12B) Film substratepreparation and polymer film synthesis by UV polymerization; (FIG. 12C)AFM height images of polyacrylic acid films crosslinked withdivinylbenzene (AA-co-DVB) with and without spherical nanoporestemplated with colloidal silica of various sizes. The average pore sizeis (from left to right) none, 15 nm, 40 nm, 120 nm, and 300 nm. Thescale bar is 200 nm. The data scale in height is (from left to right) 50nm, 50 nm, 50 nm, 100 nm, and 400 nm.

Polymer films with hexagonal pores (FIG. 13A) were also prepared by NpILfollowing a similar procedure, templated with iron oxide magneticnanocrystals with well-defined facets (FIG. 13B). Nanopores of variousother shapes can be achieved by NpIL. In an alternative approach, atop-down fabrication of square nanoposts on a silicon wafer was explored(FIG. 13D) by Achromatic Interference Lithography (AIL) for templatingsquare nanopores (FIG. 13C). AIL offers efficient patterning over alarge area with sharply delineated features (minimum radius of curvature<5 nm). The resulting square pores in the polymer film are comparable tothe spherical ones in width and depth (FIG. 13E), which enablesunambiguous differentiation of the effects of pore shape on crystalnucleation.

In FIG. 13: Angular nanopores on AA-co-DVB polymer films and theirtemplates; (FIG. 13A) AFM height image of hexagonal nanopores on thepolymer surface templated with iron oxide magnetic nanocrystals viaNpIL. The scale bar is 50 nm. (Inset) Higher resolution image of ahexagonal nanopore. The scale bar is 10 nm (FIG. 13B) TEM image of ironoxide magnetic nanocrystals as synthesized. The scale bar is 50 nm;(FIG. 13C) AFM height image of square nanopores on the polymer surfacetemplated with Si square posts. The scale bar is 200 nm; (FIG. 13D) Highresolution SEM image of Si square posts on Si wafer fabricated by AILfor templating square pores. The scale bar is 200 nm. (FIG. 13E) Depthprofiles of square and spherical nanopores of similar sizes. The scalebar is 200 nm. The square pores are 125 nm in width, 48 nm in depth, andthe spherical pores are 120 nm wide, 45 nm deep on average.

The effect of nanopatterned polymer films on the kinetics of nucleationfrom solution was quantified by measuring the nucleation induction timeof aspirin, a representative small organic molecule. The polymer filmwas made from acrylic acid crosslinked with divinylbenzene (AA-co-DVB),with which aspirin could interact via hydrogen bonding. Polymercrosslinking was designed to avoid solvent uptake and to maintain thesurface morphology when in contact with the solution. Due to thestochastic nature of nucleation events, 20 to 50 samples were testedsimultaneously to obtain the probability distribution for the nucleationinduction time. The average induction time, τ, was determined from astatistical analysis on the induction time data assuming that nucleationfollows a Poisson distribution, P(t)=exp(−t/τ). The nucleation rate, J,was calculated from τ via J=1/τV, where V is the volume of solution,with the assumption that the time scale of nucleation is much longerthan that of crystal growth, valid for this system.

In FIG. 14: Effect of the nanopore shape in AA-co-DVB polymer films onthe nucleation kinetics of aspirin: flat vs. spherical pores (left);spherical pores vs. hexagonal pores and square pores of the same size(right). J is the nucleation rate, in number of nuclei per ml ofsolution per hour, calculated from the average nucleation induction timeτ by J=1/τV, where V is the volume of solution. The standard errors of Jwere calculated from the regression on the induction time probabilitydistribution following the Poisson distribution.

As shown in FIG. 14, increasing the surface roughness by modifying thenonporous film with spherical nanopores surprisingly inhibitednucleation. The size of the spherical nanopores appeared to have littleeffect on the nucleation kinetics, within the range tested, butnucleation was promoted when angular pores of the same size were used,as shown in two cases. With hexagonal pores, the polymer film enhancedaspirin nucleation rates by more than an order of magnitude relative tothose observed with spherical pores, while in the case of square pores,a three-fold enhancement was observed. These results indicate that theledges and/or corners that distinguish angular from spherical poresacted as nucleation sites, in this embodiment, which was verified viaAtomic Force Microscopy (AFM) and X-ray Diffraction (XRD), as discussedlater.

These observations may be interpreted in terms of recent computationalresults. Simulations have shown that freezing of optimum wedge anglecorresponds to an intrinsic angle within the crystal, formed by twoclose-packed planes, at which the crystal can grow defect-free alongboth sides of the wedge. This scenario can be considered as a case ofangle-directed epitaxy, where an angle characteristic of the topologicalfeature on the substrate directs the crystal nucleation in aminimum-stress configuration, exhibited as a geometrical match betweenthe substrate and the crystal.

Angle-directed epitaxy is a possible mechanism in this case because theaspirin crystal possesses intrinsic angles formed by close-packed,low-index facets close to the characteristic angles in the nanoporestested (FIGS. 15C, 15D, and 15F). In the square nanopore, the ledge atthe intersection of the pore wall and the pore floor (L_(wf), itsdihedral angle abbreviated as a) could induce the growth of either (011)and (100), or (002) and (100) facets of aspirin ((011)

(100) or (002)

(100), with dihedral angles abbreviated as θ₀₁₁

₁₀₀ and θ₀₀₂

₁₀₀, respectively) (FIGS. 15C and 15D), where (100), (011) and (002) arethe three major facets of aspirin crystallized from bulk solution. Toestimate the extent of angular epitaxy, the cross-section of the squarenanopore was examined via High Resolution Scanning Electron Microscopy(HRSEM), and α was measured to be 96±7° in one corner of thecross-section and 101±5° in the other. This asymmetry was consistentthrough the cross-section, which may have arisen from the asymmetricstress applied to the polymer film during the template liftoff. Bothθ₀₁₁

₁₀₀ and θ₀₀₂

₁₀₀ fall in the vicinity of the smaller α, 96±7°, with θ₀₀₂

₁₀₀ being the closer match (θ₀₀₂

₁₀₀=95.84°, θ₀₀₂

₁₀₀=92.94°). Specifically, about 30% of pores contained an angle α ofwithin 1° of θ₀₀₂

₁₀₀, and around 8% within 1° of θ₀₀₂

₁₀₀. If angle-directed epitaxy were the only factor dictatingnanopore-induced nucleation, (002)

(100) would be nucleated from the ledge within the pore. The AFM imagesof aspirin crystals grown from the pores suggest it was the (011)

(100) facets that emanated from the ledge, whereas the (002) facet wasnot in contact with the pore surface (FIGS. 15A and 15B). A layeredgrowth mode is evident in both the crystal grown out from the pore (FIG.15A) and the crystals contained in the pore (FIG. 15B), which originatesfrom the aspirin dimerization via the carboxyl group within the layer,and a much weaker Van de Waals interaction between the layers. FIG. 15Bshows that these crystal layers seem to extend from the pore wall withwhich the (011) face is in contact. Moreover, the layer extensiondirection is consistent in all pores containing crystals, indicatingnucleation occurs predominantly from one side of the pore. In addition,only a fraction of the pores induced nucleation. These observationsprovide evidence that the (011)

(100) and not (002)

(100) facets were nucleated from L_(wf), but may be from those ledgeswith the appropriate angle α. These growth patterns can be attributed tothe favorable interactions between (011)

(100) and the polymer surface, as inferred from the characteristicfunctionalities displayed on their respective surfaces (FIGS. 15C and15E). (011) and (100), rich in carboxyl and carbonyl groups, can formhydrogen bonds with the carboxyl groups on the AA-co-DVB polymersurface, whereas the nonpolar (002) plane may interact with the polymermuch more weakly. This result suggests that solute-polymer interactionscan play an important role in determining nucleation behavior in angularpores, in addition to angle-directed epitaxy.

In FIG. 15: Angle-directed epitaxy of aspirin crystals induced byangular nanopores; (FIG. 15A) AFM phase image of aspirin crystals grownout from the square pores; (FIG. 15B) AFM phase image showing (100)layers of aspirin crystals nucleated at ledges in the square pores. Thescale bar is 100 nm; (FIG. 15C, FIG. 15D) Representative epitaxyconfigurations of aspirin crystal facets along the ledge of the squarepore; (FIG. 15E) Proposed aspirin-polymer interactions at thecrystal-polymer interface; (FIG. 15F) Proposed epitaxy configuration ofaspirin crystal facets at the corner of an hexagonal pore; (FIG. 15G,FIG. 15H) AFM phase image of an aspirin crystallite grown from the 15 nmhexagonal pores and its possible orientation; (FIG. 15I) AFM heightimage of the surface of an aspirin crystal grown on and detached fromthe AA-co-DVB polymer film with hexagonal pores.

Following the principles of angle-directed epitaxy assisted by favorableinteractions of the crystal facets with the substrates, the cornerswithin hexagonal pores may act as nucleation sites to induce the growthof (011)

(011)

(100) with (100) in contact with the pore floor, and (011)

(011) with the pore walls (FIG. 15F). This may be because the anglemismatch is very small in this configuration, and all three faces ofaspirin could interact with the polymer surface via hydrogen bonding. Ifnucleation ensued from the corner, the growth thereafter may haveresulted in an aspirin crystallite that fit comfortably inside the poreand took on the shape of a hexagon, given that other intrinsic angles ofthe crystal also matched quite well with the pore geometry (FIG. 15H).Indeed, crystallites with comparable shape and size to those of the porewere observed via AFM on the surface of aspirin crystals detached fromthe polymer film (FIGS. 15G and 15I). In addition, XRD results verifiedthat the (100) face was in contact with the pore floor. Theseobservations support corner-induced nucleation from hexagonal pores inthis embodiment.

Based on the experimental and computational evidence, a molecularmechanism to interpret the pore shape effect on nucleation is nowdescribed. Crystal nucleation from solution is preceded by molecularcluster formation via density fluctuations and molecular re-orientationthrough structure fluctuations; both may be required for nucleation. Therate of nucleation can be modified in two ways by the presence of anamorphous, nanoporous surface in a metastable solution. First, favorablesurface-solute interactions can enrich solute concentrations near thesurface, facilitating molecular cluster formation. Second,ledges/corners in the pore can induce partial orientational order of thesolute in domains close to the surface via specific interactions andgeometrical confinement, which enhances the solute molecularrealignment. When the molecular orientation imposed by the ledge/cornergeometry resembles that in the crystal, the rate of nucleation isincreased to the greatest extent, the macroscopic expression of which isangle-directed epitaxy. This can also implies that for angular nanoporesto promote nucleation, favorable surface-solute interaction may berequired, in some embodiments. To verify this point, the chemical makeupof the polymer film was altered from AA-co-DVB to AM-co-DVB (FIG. 16).This chemistry was selected out of the polymer films tested because, inthe absence of pores, it exhibited no effect on aspirin nucleation frombutyl acetate, indicating that surface-solute interactions are notsufficiently strong to affect nucleation under these conditions.Patterning of the AM-co-DVB surface with the same angular nanopores didnot lead to enhanced nucleation kinetics relative to nucleation onnonporous films (FIG. 16).

In FIG. 16: Effect of polymer surface chemistry on kinetics of angularnanopore-induced nucleation of aspirin: AA-co-DVB vs. AM-co-DVB. J isthe nucleation rate, in number of nuclei per ml of solution per hour. AMdenotes 4-acryloylmorpholine. AA denotes acrylic acid. AM-co-DVB refersto poly 4-acryloylmorpholine crosslinked with divinylbenzene.

In summary, nanopore shape can play a key role in determining thekinetics of surface-induced nucleation probed with a small organic modelcompound. Angular pores of chosen geometry and surface chemistrypromoted nucleation, while spherical pores of the same size did not inthis example. Ledges in the square pores and corners in the hexagonalpores were active nucleation sites, following the principle ofangle-directed epitaxy. The pore geometry and specific surface-soluteinteractions may jointly determine which crystal facets would nucleatepreferentially. Favorable surface-solute interactions may be requiredfor angular pores to promote nucleation. A molecular mechanism may bepresent by which the pore shape affects nucleation by altering themolecular orientational order near the ledges/corners of the pores.

Methods

Fabrication of Polymer Films with Spherical Nanopores:

Quartz slides (75 mm×25 mm) were treated with O₂ plasma to enrich thesurface in hydroxyl groups. Two hundred microliters of 5 w % colloidalsilica (commercially available) were spread on the quartz slide andallowed to self-assemble during slow water evaporation over 12 hours.The self-assembled SiO₂ and the quartz slide were then sintered at 800°C. for 5 min to coalesce the particles with the quartz slide and formthe imprint mold. The film substrate (25 mm×5 mm) was prepared bytreating a glass slide with O₂ plasma followed by silanization withtrichlorosilane in a vacuum oven at 40° C. Silanization is necessary toadhere the polymer film to the substrate and avoid film cracking andpeeling. One microliter prepolymer mixture of monomer acrylic acid (AA),crosslinker divinylbenzene (DVB), and initiator IRGACURE 2022 weresandwiched between the imprint mold and the film substrate. The molarratio of monomer to DVB was 2:1. The concentration of IRGACURE 2022 was4 v % with respect to DVB. The prepolymer mixture was then polymerizedunder UV irradiation for 15 min, at 72 mW/cm². After irradiation, theimprint mold was peeled off and the polymer films were annealed at 70°C. in a vacuum oven for 3 h to remove unreacted species. Parts weregenerally pre-cleaned and assembled in a Bio-safety cabinet to reducecontamination by impurities, which may interfere with polymer filminduced nucleation.

Fabrication of Polymer Films with Angular Nanopores:

Polymer films with hexagonal nanopores were synthesized following aprocedure similar to that described above, templated with iron oxidemagnetic nanoparticles (MNP). The presence of sufficient surfactants(oleic acid) during synthesis was important to obtain sharply definedfacets of MNP crystals. As in the case of making spherical nanopores,colloidal self-assembly was utilized for preparing the imprint mold,which was made by spreading 20 μl MNP-decane solution (˜9 w %) on aplasma cleaned quartz slide (75 mm×25 mm) and allowing the decane toevaporate over a period of 6 hours. The excessive surfactants present inthe nanocrystal dispersion also participated in the assembly processleaving space for polymers to form between the nanocrystals. Afterpolymerization, the imprint mold was peeled off from the polymer film,the nanocrystals on the film were subsequently dissolved with dilutehydrocholoric acid (˜1 N), and the film was rinsed with deionized waterfollowed by acetone and vacuum drying. The imprint mold for makingsquare pores was fabricated by Achromatic Interference Lithography atthe MIT Research Laboratory of Electronics. The mold took the form of120 nm Si square pillar arrays with 200 nm pitches covering a 3-inch Siwafer. The top edges of the pillars were sharply defined with radii ofcurvature less than 5 nm. Large area patterning is necessary for makingsufficient copies of polymer films to obtain the induction timeprobability distribution. The polymer film synthesis and post-processingprocedures were the same as those used in the preparation of sphericalnanopores. The effects of polymer films with pores on nucleationkinetics were compared against those in the absence of pores, which weresynthesized following the same procedure with the quartz surface as withthe template.

Nucleation Induction Time Measurement:

Once synthesized, the polymer film with its substrate was insertedvertically into a 1 ml glass shell vial containing 200 μl 47 mg/mlaspirin solution in butyl acetate. For each polymer sample, 20-50 vialswere assembled and immersed in a circulator stabilized at 50±0.1° C. todissolve any pre-existing crystals, and then the solution was quenchcooled to 5±0.1° C. by immersing into a second circulator. The number ofvials in which crystallization occurred was recorded as a function oftime. All the operations involving exposing polymer films, aspirinsolutions and shell vials to the atmosphere were conducted inside a BioSafety Cabinet to reduce impurity contamination to the lowest level.Efforts were made to clean all components before usage and aspirinsolutions were filtered with an Acrodisc 0.2 μm PTFE syringe filter.

Characterization:

Atomic Force Microscopy (AFM) and Powder X-ray Diffraction (XRD) wereemployed to study the aspirin crystal orientation inside the angularnanopores on the polymer films after the nucleation induction timestudy. AFM images were obtained with a Dimension 3100 XY closed loopscanner (Nanoscope IV, VEECO) equipped with NanoMan software. Height andphase images were obtained in tapping mode in ambient air with silicontips (VEECO). The crystal orientation was verified with XRD to identifythe specific crystallographic planes parallel to the polymer film. TheX-ray diffraction patterns were recorded with a PANalytical X'Pert PROTheta/Theta Powder X-Ray Diffraction System with Cu tube and X'Celeratorhigh-speed detector. No less than five polymer films were examined withXRD on each type of polymer sample.

Synthesis of Faceted Fe₃O₄ Nanoparticles:

Materials: Iron tri(acetylacetonate) (Fe(acac)₃) (97%),1,2-tetradecanediol (90%), oleic acid (OA) (90%), and benzyl ether (99%)were purchased from Sigma Aldrich. n-Decane (99%) was purchased fromAlfa Aesar. Methanol (99.8%) was purchased from Mellinkrod. Allchemicals were used as received. All water utilized in the experimentswas Milli-Q (Millipore) deionized water. Synthesis method: Colloidaldispersions of faceted Fe₃O₄ nanoparticles were prepared by a slightlymodified procedure of these method for the synthesis of spherical Fe₃O₄nanoparticles reported previously (e.g., see Harada, T.; Hatton, T. A.Langmuir 2009, 25, 6407). In brief, iron tri(acetylacetonate) (2 mmol),1,2-tetradecanediol (10 mmol), oleic acid (12 mmol), and benzyl ether(20 mL) were mixed and stirred magnetically under flowing nitrogen. Themixture was heated gradually to 200° C. and kept at this temperature for2 h. Then, the temperature was increased up to the reflux condition(300° C.) under a blanket of nitrogen, and kept for 1 h at reflux. Theblack reacted liquid was cooled to room temperature by air-cooling andtransferred from the reaction flask to a centrifugation bottle. Onaddition of methanol (40 mL) to the reaction mixture, the blacknanoparticles precipitated and were separated via centrifugation (9000rpm, 10 min). To remove the residual reacting materials, theprecipitated nanoparticles were rinsed with methanol several times.After the precipitated nanoparticles were well-dried, 10 mL of n-decanewas added to the precipitate and the mixtures were ultrasonicated.

Nucleation Induction Time Study to Select Polymer Chemistry:

In this study, polymers with smooth surfaces were synthesized directlyin the glass shell vials used for crystallization, instead of on a glasssubstrate as in the case of polymer films with nanopores. In this way,the impurities and active nucleation sites from the glass substrateswere substantially eliminated.

30 μl prepolymer mixture of monomer, crosslinker divinylbenzene (DVB),initiator IRGACURE 2022 were injected into the 1 ml pre-cleaned glassshell vials under the Bio-Safety Cabinet. The monomers tested were4-acryloylmorpholine (AM), 4-Hydroxybutyl acrylate (HBA), and acrylicacid (AA). The molar ratio of monomer to DVB was 2:1. The concentrationof IRGACURE 2022 was 4 v % with respect to DVB. The prepolymer mixturewas subsequently polymerized under UV irradiation under N₂ protectionfor 30 min, at ˜10 mW/cm². Less UV intensity and longer irradiation timewere applied to avoid polymer cracking after synthesis. Afterpolymerization, the polymers and the shell vials were allowed to slowlycooled down for 30 min before annealed at 70° C. in a vacuum oven for 5h to remove unreacted species.

The nucleation induction time study followed the same procedure asstated in the methods section. In some cases, the bottom of the vialswere attached 3/8-inch thick rubbers to block heat transfer from thebottom of the vials. This may be necessary in some cases because thepolymers conformed to the bottom of the vials differ in heatconductivity, resulting in different cooling rate in the solution if theheat transfer were allowed through the bottom of the vial, and thecooling rate significantly impacts the nucleation induction time. Inaddition, enough vial spacing was designed to allow equivalent coolingrate around each vial.

Supplementary Results and Discussion

Crystal Orientation by XRD:

In FIG. 17: X-ray diffraction pattern of aspirin crystals grew from thebutyl acetate bulk solution (top), on AA-co-DVB films with 125 nm squarenanopores (middle), and on AA-co-DVB films with 15 nm hexagonalnanopores (bottom).

In this embodiment, as shown in FIG. 17, (100) facet of aspirin wasidentified as the preferred orientation on AA-co-DVB films with angularpores, relative to the random orientation of bulk crystals. This resultindicates that (100) facets grew parallel to the polymer surface, whichis in line with the observation by AFM (FIG. 15) and the inference fromangle-directed epitaxy and favorable polymer-solute interactions. TheXRD pattern in the case of square pores also suggests that a fraction ofcrystals grew with (011) facet parallel to the polymer surface. It ismade possible when aspirin nucleates from ledge inside the pore with(011) facet in contact with the pore floor and (100) with the pore wall,which also satisfies the rules of angle-directed epitaxy assisted withfavorable polymer-solute interactions.

Assignment of Aspirin Crystal Facets:

The crystal facets in the AFM images (FIG. 15) were assigned based onthe following observations. First, a layered growth mode is evident fromboth the overgrown crystal (FIG. 15A) and the crystals contained in thepore (FIG. 15B), which originates from the aspirin dimerization via thecarboxyl group within the layer, and a much weaker Van de Waalsinteraction between the layers. It is these layers that constitute theaspirin (100) plane, which was confirmed by XRD that the (100) planegrew parallel to the film surface (FIG. 17). Second, the terrace formedby the edges of developing layers points towards the crystal growthdirection (FIG. 15A), which is along the (010) axis, and the slowgrowing face (002) is left to define the crystal edges. This assignmentis in agreement with the dihedral angles exhibited by the crystals inthe pore (FIGS. 15A and 15B).

Nucleation Inhibition by Spherical Nanopores:

Monte Carlo simulation have shown that freezing of hard sphere colloidsare frustrated on curved surfaces, on which crystals cannot grow free ofstress, and the resulting defects increased the nucleation barrier tonucleation. Concave surfaces with radius of curvature (R_(S)) from 10 to150 times the diameter of the colloids (a), which overlaps with therange that was investigated (R_(S)=12.5, 33, 100σ; σ, the size ofaspirin, is 6 Å estimated from the crystal density were previouslyinvestigated). However, this trend was generally not observed,suggesting in this study that the nucleation on a curved surface becomesincreasingly difficult on a more strongly curved surface. It may be due,at least in part, to the practical limitations in the experiment.Particularly, it is very hard to eliminate unintended nucleation sitesfrom the system, either on the film substrate or residue dust particles,which could trigger nucleation and prevent measurement of the realextent of nucleation inhibition by the spherical pores. It is evidencedby the observation that crystals mostly precipitated from elsewhere inthe system, rather than grew out from the film with spherical pores.Attempts were made to avoid and remove as much impurities as possiblefrom the system and to silence the nucleation sites on the filmsubstrate by coating or functionalizing with inert moieties, but theaverage nucleation induction time could not be further lengthened.Nonetheless, the observation is largely in line with the implicationfrom the computational study, and the inhibition effect by the sphericalpores is most likely greater than experimentally observed. In contrastto simulations, the inhibition effect of nanopatterned surfaces wasmeasured against imperfect flat surfaces in practice. Thus thenucleation inhibition could also be attributed to the elimination ofactive nucleation sites from the flat surface by replacing them withinactive spherical nanopores.

Comparison of Polymer Chemistry Effect on Aspirin Nucleation:

In FIG. 18: Role of surface chemistry in nanopore-induced nucleation;Statistical analysis of aspirin nucleation induction times with andwithout nonporous polymers. The linear regression follows the formulaln(P)=−t/τ to obtain the average induction time τ. P is the probabilityfor no crystallization event to occur within time t. The standard errorof τ was obtained from linear regression of the slope.

FIG. 18 illustrates the polymer chemistry effect on aspirin nucleation.Compared with the bulk, polyacrylic acid crosslinked with DVB(AA-co-DVB) showed marked effect on aspirin nucleation kinetics,yielding an average induction time consistent with that observed withnonporous AA-co-DVB films on glass substrate (FIG. 14 left). Incontrast, HBA-co-DVB slightly affected nucleation, and AM-co-DVBvirtually no effect. Thus, AM-co-DVB was chosen as a negative control todemonstrate the effect of polymer chemistry on theangular-nanopore-induced nucleation (FIG. 16). The observed averagenucleation induction times with polymer films on the glass substrate(FIG. 16) were much shorter than that measured with polymer surfaceswithout substrates because of the interference from the activenucleation sites on the substrate and the impurities brought in by thesubstrates, aforementioned.

The effect of surface chemistry on nucleation kinetics could result froma competition between the solute-polymer interaction and thesolute-solvent interaction. Specifically, for aspirin molecules tonucleate on the polymer surface, aspirin needs enrich near the polymersurface driven by specific solute-polymer interactions, however, suchinteraction is shield by the solvent-solute interactions to certainextent. In the case of AM-co-DVB, the carbonyl group of solvent butylacetate could complete with that of the polymer to interact with thecarboxyl group of aspirin via hydrogen bonding, imposing strongshielding effect; as for AA-co-DVB, it can form carboxylic dimers withaspirin molecules, with which the strength of butyl acetate-aspirininteraction might not complete with.

Comments on the Nucleation Activities of Hexagonal Pores Vs. SquarePores:

The observed difference in nucleation activities of hexagonal pores vs.square pores may arise from several factors. First, the polymer filmswith hexagonal pores have higher pore area density (Table 3), which mayhave contributed to its higher nucleation activity. However, the areadensity of pores is not equivalent to the area density of activenucleation sites. Specifically, corners in the hexagonal pores are mostlikely to be the nucleation sites, whereas in the case of square pores,ledges are identified to be responsible for induction aspirinnucleation. One-dimensional nucleation sites (corners) cannot bedirectly compared with two-dimensional ones (ledges) in terms of thearea density. Second, aspirin grows from the corners in hexagonal poreswith three facets in contact with the pore wall, which is likely to bemore energetically favorable compared with nucleation from ledges wheretwo out of these three facets are in contact with the ledge planes. Tofurther the investigation, nanopores of various geometries with the samesize will be fabricated and molecular dynamic simulation is beingperformed to gain insight.

TABLE 3 Nucleation induction time of aspirin induced by AA-co-DVB filmspatterned with angular nanopores. Pore shape Hexagonal Square Pore size15 nm 125 nm τ (min) 26 ± 7 138 ± 12 # of pores/μm² 700 25 # ofcorners/μm² 4200 100

Example 4

This Example described the role of polymer-solute interactions ingel-induced nucleation.

Introduction:

Interfaces present in a metastable liquid are believed to have aprofound impact on nucleation behavior. Considerable strides have beenmade over the last few decades towards understanding the effect ofinterfaces on nucleation and several mechanisms have been proposed. Theepitaxy mechanism has been established to describe crystal formation oncrystalline surfaces or surfaces with two-dimensional symmetry. Surfacesmay also affect nucleation via polarization matching with thecrystallizing molecule when both the surface and the crystal exhibit netdipole across the surface/crystal interface. These mechanisticunderstanding should provide guidance for designing surfaces to controlcrystal nucleation. However, the applicability of these approaches isrestricted to a large extent, because the surface properties involvedmay not be adjustable catering to the system of interest, and one may belimited to surfaces with 3D or 2D symmetry, such as crystal facets,self-assembled monolayers, and Langmuir-Blodgett films, etc.Non-crystalline polymeric materials offer a promising alternative, whosestructure, topology and chemistry are easily tunable over a wide rangeby a variety of established fabrication methods. Particularly, polymergels with tunable microstructures can aid in controlling nucleationkinetics. Polymer gels are unique in their ability to concentrate solutemolecules via thermodynamic partitioning driven by favorablepolymer-solute interactions, and intermolecular interactions may aid inpromoting nucleation.

Intermolecular interactions have been demonstrated to play an importantrole in dictating the nucleation behavior at interfaces. However,mechanistic understanding is still insufficient to enable rationaldesign of surface chemistry for controlling nucleation of molecularcrystals from solution. The complexity partially arises from weakintermolecular interactions in molecular systems relative to ionic,metallic, and/or covalent crystals, flexible molecular conformations,and/or intricate solvent effects. In practice, the influence ofintermolecular interactions on nucleation is often convoluted with otherfactors such as surface lattice structures, surface morphology, etc.,making it more challenging to study.

This example helps to elucidate the role of intermolecular interactionsin gel-induced nucleation and its interplay with the effect of polymermicrostructures on nucleation. Chemically modified polymer microgels viacopolymerization were synthesized and were studied and were studied itseffect on nucleation kinetics as compared to unmodified microgels.Nucleation kinetics of model compounds was very sensitive to thepolymer-solute interactions, and dramatic acceleration of nucleation wasobserved when the strength of polymer-solute interactions was increasedmarkedly. The functionalized microgels left signatures on nucleationinduction time distribution, in some embodiments, featuring twocharacteristic time scales, which may suggest chemical heterogeneity atnanometer scale due to copolymerization. The underlying mechanism fromthe perspective of adsorptive partitioning and templating effect wasexplored to interpret the role of intermolecular interactions ingel-induced nucleation.

Results and Discussion

Synthesis of the Polymer Microgels:

Two model polymer chemistries were chosen for synthesis of microgelparticles to use in gel-induced nucleation studies. The first werecrosslinked homopolymer gels of poly(ethylene glycol) diacrylate(PEG_(M)DA) of various monomer molecular weight, M (g/mol). The secondwere co-polymers of PEGDA and 4-acryloylmorpholine (AM). AM was selectedas a co-monomer to functionalize the PEGDA gel because it containsmultiple hydrogen-bond acceptors, which may interact favorably with thehydrogen-bond donors of aspirin (ASA) and acetaminophen (ACM), the modelcompounds employed in this example.

Synthesis of model microgel PEGDA and PEGDA-co-AM microgels was carriedout using Stop Flow Lithography as described in herein. Cube-shapedmicrogels were prepared to facilitate imagining such that theorientation of surface-attached crystals was unambiguous.

PEGDA microgels were prepared from a range of monomers with M=130-700g/mol using pre-cursor fluids containing a fixed concentration of PEGDAof 25 vol %. Similarly, PEGDA-co-AM microgels were prepared using thesame range of monomer molecular weights containing 15 vol % PEGDA and 15vol % AM. The range of PEGDA molecular weights thus represents a rangeof crosslinking density across the different microgel particles,resulting in a range of the average mesh size, ξ, of the crosslinkedgel; i.e., the average distance between crosslinks within the polymernetwork. The particular pre-cursor concentrations of PEGDA and AM usedwere chosen to closely match ξ between the two systems in order toisolate the effect of polymer chemistry on nucleation kinetics.

Characterization of Polymer Microstructure:

The microstructure of PEGDA and PEGDA-co-AM gels was characterized inorder to better elucidate the nature of polymer-API interactions andtheir effect on nucleation. Estimates of were obtained from equilibriumswelling measurements using a procedure described previously. FIG. 19compares the apparent mesh size from swelling measurements (closedsymbols) obtained previously measured for PEGDA microgels (i) to thatobtained for PEGDA-co-AM microgels (ii) with increasing M. Theincorporation of AM into the PEGDA hydrogel network resulted in a mildincrease in mesh size on the order of 10-25% over the range of PEGDAmolecular weights studied. This is expected, since the effectivelengthening of the acrylic polymer backbone by insertion of AM monomersis small compared to the overall length of PEG chains.

In FIG. 19: Mesh size of PEGDA (i) and PEGDA-co-AM (ii) hydrogelsmeasured in 38/62 (v/v) ethanol/water at 23° C. using estimated byequilibrium swelling measurements (closed symbols) and SANS analysis(open symbols).

In order to examine the microstructure of the PEGDA and PEGDA-co-AM gelsin further detail, as well as to validate several assumptions made inthe equilibrium swelling measurements, small angle neutron scattering(SANS) measurements were performed on representative hydrogel sampleswith M=200 g/mol and 700 g/mol. The corresponding absolute intensityspectra, I(q)−I_(bk) are shown in FIG. 20, where the incoherentbackground intensity, I_(bk), has been subtracted. The data were fit toa generalization of the Debye-Bueche model:

$\begin{matrix}{{I(q)} = {\frac{A}{1 + \left( {\xi \; q} \right)^{m}} + \frac{B}{\left\lbrack {1 + \left( {\Xi \; q} \right)^{n}} \right\rbrack^{2}} + {I_{bk}.}}} & (1)\end{matrix}$

The first term is used to describe local fluctuations of individualchains with excluded volume constrained by crosslinks, whose lengthscale is set by the mesh size, ξ. The scaling exponent m is related tothe solvent quality of the polymer chains; e.g., m=2 for Gaussianchains, whereas m<2 for chains in a good solvent. The second termdescribes the low-q structure, and arises from large-scale heterogeneity(either static or dynamic) within the material, where Ξ is thecharacteristic length scale of structural inhomogeneity. The scalingexponent n is related to the nature of the interface betweeninhomogeneous regions of the material. It is typically assumed that n=2,corresponding to sharp interfaces between inhomogeneities. Thisrestrictive assumption generally resulted in poor fits to the SANS datacollected for both PEGDA and PEGDA-co-AM microgels. Therefore, theDebye-Bueche model were generalized by leaving n as an adjustableparameter. This is empirically equivalent to assuming that the densityprofile between homogeneities can be described by scattering with asurface fractal dimension of n²; i.e., n²=4 for a sharp interface,whereas 3<n²<4 for a diffuse interface.

In FIG. 20: Absolute SANS intensity spectra for the polymer hydrogelsindicated. Solid lines give best fits to the Debye-Bueche model Eq. (1).

TABLE 4 Structural properties of PEGDA and PEGDA-co-AM hydrogels fromSANS analysis. Polymer M_(n)(g/mol) ξ (nm) m Ξ (nm) n PEGDA 200 0.92 ±0.06 1.88 23.3 ± 1.4 1.88 700 2.09 ± 0.08 1.59 53.6 ± 1.4 2.16PEGDA-co-AM 200 1.05 ± 0.06 1.38 10.4 ± 1.6 1.80 700 2.39 ± 0.08 1.3461.6 ± 1.2 2.04

Eq. (1) was fit to the experimental data, and the best-fit modelpredictions are shown in FIG. 20, with the corresponding modelparameters are listed in Table 4. The generalized Debye-Bueche modelgave a quantitatively accurate description of the data. Thus, themicrostructure of both PEGDA and PEGDA-co-AM microgels exhibitedsignificant structural heterogeneity over length scales ranging from10-60 nm. The length scale for heterogeneity, given by Ξ, ranges from10-20ξ for the PEG₂₀₀DA polymers, and decreases upon addition of the AMco-monomer. By contrast, Ξ is approximately equal for both the PEG₇₀₀DAhomopolymer and its AM co-polymer, with a value that of Ξ˜25ξ.Furthermore, the Porod exponent n˜2 for the PEG₇₀₀DA gels suggest sharpinterfaces between structural inhomogeneities, whereas n˜1.8-1.9 for thePEG₂₀₀DA samples suggests a transition to more diffuse interfaces at lowPEGDA molecular weight.

Although the nature of this heterogeneity is unknown, such structuretypically arises from microphase separation within the hydrogel, wherethe structure exhibits distinct regions of different density. For thePEGDA and PEGDA-co-AM gels studied here, the phase separation couldeither be between the polymer and solvent, between the variousconstituent moieties of the polymer (ethylene glycol, acrylate, and AM),or a combination of both phenomena. For example, previous studies haveshown that formation of PEGDA hydrogels in the presence of highmolecular weight PEG porogens leads to polymer phase separation and theformation of micron-scale pores within the hydrogel. However, opticalimaging of the microgel particles considered here exhibited no evidenceof such large-scale porosity. Turning to the SANS results, note thatboth and n are found to primarily depend on the PEGDA monomer molecularweight, and not the presence of AM co-monomer. Since the primarychemical difference between the PEG₂₀₀DA and PEG₇₀₀DA monomers is therelative amount of acrylic groups compared to ethylene glycol units,therefore the structural heterogeneity within the hydrogels may bedriven by microphase separation of the polymerized acrylic groups.

In FIG. 21: Schematics of microgel structures inferred from SANSmeasurements. Long light grey chains (e.g., i), short black chains, andshort dark grey chains (e.g., ii) denote the PEG subchain, acrylate andAM segments, respectively.

FIG. 21 shows diagrams of possible structures for the PEGDA (top) andPEGDA-co-AM (bottom) hydrogels under such a scenario. In the so-calledreaction bath in which the crosslinked network is formed (left), thenascent hydrogel exhibits homogeneous microstructure. At equilibrium(right), however, phase separation of the acrylic backbone chains leadsto phase separation, where acrylate-rich regions coexist withacrylate-poor regions. This depiction of the microstructure isconsistent with the observed trends in SANS data, as follows. Since thepoly(ethylene glycol) strands of the gel is attached at the ends byacrylic groups, the length scale Ξ will be primarily determined by thelength of PEG chains (blue) between neighboring acrylic backbone chains(red). This explains the observed trend in Ξ, which increases for 3-5fold as the PEGDA molecular weight is increased from 200 g/mol to 700g/mol. Since the addition of AM co-monomer (green) within the gelgenerally occur along the acrylic backbone chains, the AM groups willthus primarily be contained within the AM-rich regions. This explainsthe fact that neither Ξ nor n change significantly uponco-polymerization with AM, since the AM groups does not significantlyaffect the structure of the AM-poor regions.

Regarding the smaller length scale structure of the gels, given by themesh size and free volume exponent m, given the previous discussion, thedefinition of a uniform average mesh size, such as that obtained fromswelling measurements, is insufficiently describe the microstructure ofthe microgels. The measured values of from the SANS measurements aregenerally in fair quantitative agreement with those measured byequilibrium swelling measurements (FIG. 19). It is reasonable to presumethat averaging of the mesh size over various polymer-rich andpolymer-lean regions within the gel may result in an average mesh sizethat is similar to that measured in a macroscopically-averagedmeasurement such as swelling.

In contrast to the large-scale heterogeneous structure, m dependedsignificantly on the presence of AM co-monomers within the hydrogel. Forthe PEGDA homopolymer gels, m˜1.6-1.8, indicating that the polymerexhibits behavior characteristic of flexible chains in a good solvent,as expected for PEG in aqueous solution. By contrast, m˜1.3-1.4 for thePEGDA-co-AM co-polymer gels. This value of m is significantly outsidethe range of 5/3<m<2 expected for flexible chains in a good solvent, andin the range of 1<m<1.5 expected for semi-flexible chains. Although thesource of such behavior is unclear, one possible explanation is a changein stiffness of the acrylic backbone chains upon co-polymerization ofthe bulky AM co-monomers, resulting in an overall decrease inflexibility of the polymer at length scales less than the mesh size.

Quantification of Polymer-Solute Interactions:

The strength of intermolecular interactions between the PEGDA-co-AMpolymer network and the molecule to crystallize was characterized withthe solute equilibrium partition coefficient at the same condition asused in the crystallization study. Solute partition coefficient κ,defined as the ratio of solute mass fraction in solution confined in thegel to that in the bulk, is a relevant parameter because it informs thesolute concentration in the gel at the crystallization condition, whichis an important factor affecting nucleation. Shown in FIG. 22, κ of ASAincreased by 60% on average after introducing AM into the PEGDA gel, andthe ASA concentration in the PEGDA-co-AM gels reached as high as sixtimes as that in the bulk solution. This result indicates much strongerinteractions between ASA and the polymer matrix after functionalization.Before chemical modification, κ climbed from 3.4 to around 4.2 with theincrease of M_(n), the PEG molecular weight of the PEGDA monomer, whileafter modification, κ became insensitive to M_(n). This observationsuggests that ASA mainly interacted with AM segments of PEGDA-co-AMpolymer in the solution environment, for reasons discussed as following.The PEGDA polymers are comprised of the PEG subchain and the acrylatecrosslinkers. As M_(n) increases, the mass ratio of PEG to acrylateincreases, so does κ in the case of PEGDA system, indicating that ASAprimarily associates with the PEG subchain. This inference is furthersupported by the fact that the molar ratio of ethylene oxide units inPEG to ASA remained constant (7.7) for all mesh sizes, calculated fromthe partition experiments. In the case of PEGDA-co-AM, the mass fractionof AM doesn't change with M_(n), and correspondingly, κ also turnedinvariant yielding a constant AM to ASA molar ratio of around unity.This result provides evidence that ASA preferred to interact with AMthan with PEG or acrylate groups constituting the polymer gel.

The ASA-polymer interactions were further quantified with the soluteadsorption enthalpy via Isothermal Titration calorimetry (ITC), whichalso helps to deepen the understanding of partitioning effect. FIG. 23shows the results of ITC measurements, where the enthalpy of interactionbetween ASA and both PEG₄₀₀DA and PEG₄₀₀DA-co-AM microgels is plottedversus the equilibrium concentration of ASA. The data are presented bothas instantaneous enthalpies at a given concentration, ΔH_(ASA-gel)(top), and as cumulative enthalpies up to a certain concentration,ΔH_(ASA-gel) ^(tot) (bottom). At low ASA concentrations, ΔH_(ASA-gel)exhibits a plateau for both PEGDA and PEGDA-co-AM gels. After titrationof ASA to a concentration of 10 mM or greater, ΔH_(ASA-gel) decreasesmonotonically, approaching zero at large ASA concentrations. Thisbehavior suggests that the mechanism of ASA-polymer interactions is byadsorption of ASA onto the polymer network. This is particularlyapparent when examining the cumulative interaction enthalpy,ΔH_(ASA-gel) ^(tot), which exhibits the qualitative features of anadsorption isotherm, such that ΔH_(ASA-gel) ^(tot) is related to theequilibrium surface coverage of ASA on the polymer hydrogel. At lowconcentrations, the increase of ΔH_(ASA-gel) ^(tot) with ASAconcentration is roughly linear, corresponding to ideal adsorption ofASA where a majority of the injected solute molecules adsorb to thesurface. However, at sufficiently large ASA concentrations, ΔH_(ASA-gel)^(tot) tends toward a plateau value, suggesting saturation of thehydrogel surface due to monolayer coverage of ASA. Attempts to fitsimple, one-site adsorption isotherms to the data in FIG. 23 wereunsuccessful, most likely due to the complicated structure and chemistryof the hydrogel surface. Nevertheless, the considerable range ofconcentration over which ΔH_(ASA-gel) ^(tot) increased linearly with ASAconcentration allowed for calculation of the enthalpy of adsorption ofASA at infinite dilution, ΔH_(ASA-gel) ^(∞) by averaging ΔH_(ASA-gel)over ASA concentrations in the plateau region (FIG. 23), resulting inΔH_(ASA-gel) ^(∞)=−9.8 kcal/mol for PEG₄₀₀DA and ΔH_(ASA-gel) ^(∞)=−12.3kcal/mol for PEG₄₀₀DA-co-AM. This confirms that ASA-polymer interactionswere more favorable for PEGDA-co-AM hydrogels compared to PEGDAhydrogels, and further suggests that the presence of the AM co-monomerenhanced adsorption of ASA.

Compared with the ASA system, the ACM-polymer interactions turned out tobe much weaker in both the microgels (FIG. 22), indicated from lower κvalues. A marginal increase in κ was seen with modified gels, rangingfrom 35% (M_(n)=130 g/mol) to 14% (M_(n)=700 g/mol) Similar to ASA, ACMpartitioned to a similar extent into the modified gel of all mesh sizes,whereas in unmodified gels, κ exhibited more apparent variation as afunction of M_(n). This result may also imply that ACM interactsstronger with AM than with PEGDA. Comparing the ASA to ACM systems, itis not apparent why ASA interacted stronger with both the polymers thanACM. One might expect the reverse since the ACM molecule carries morehydrogen-bond donors, and both PEGDA and AM are rich in hydrogen-bondacceptors. Complimentary functional group interactions, commonlysolicited for interpreting the substrate effect on nucleation fromsolution, did not explain these observations, possibly because thisapproach does not account for the fact that both the polymer and thesolute are well solvated. Increased cost of de-solvation required forACM adsorption onto the polymer may have led to its decreasedpartitioning, since solute-solvent interactions are stronger for ACMthan for ASA indicated by higher ACM solubility in 38/62 (v/v)ethanol/water mixture (90 mg/ml at 25° C.) than that of ASA (32 mg/ml at25° C.).

In FIG. 22: Comparison of partition coefficient, κ, in the PEGDA gelsvs. PEGDA-co-AM gels for ASA (top) and ACM (bottom) systems. κ isdefined as the ratio of solute mass fraction in solution confined in thegel to that in the bulk. The error bars are calculated from three tofour independent repeats.

In FIG. 23: Enthalpy isotherms for adsorption of ASA onto PEG₄₀₀DA (opensymbols) and PEG₄₀₀DA-co-AM (closed symbols) microgels, includinginstantaneous (top) and cumulative (bottom) enthalpies of adsorption.Straight line gives fit to obtain the infinite dilution enthalpy ofadsorption. Solid lines show the region over which the infinite dilutionenthalpy of adsorption was calculated.

Effect of Polymer Gels on Nucleation Induction Time Statistics:

To evaluate the impact of polymer-solute interactions on nucleationkinetics, induction times of ASA and ACM were measured with microgels ofa series average mesh sizes before and after chemical modificationsuspended in respective supersaturated solutions. The volume fraction ofmicrogels in the solution is so small (˜10⁻⁵) that the solutepartitioning in the gels does not affect the bulk concentration. Foreach system, a large number of experiments (50-100) were conducted toobtain the induction time probability distribution. The nucleationinduction time generally follows the Poisson distribution. However,deviations can occur, as observed in this study, when there is more thanone type of nucleation sites in a sample, giving rise to multiplePoisson processes with different characteristic time scales.

For samples with PEG_(M)DA microgels, the nucleation induction timedistributions reported in previous work can be faithfully described bystretched exponentials (Tables 5 and 6), P=exp[−(t/τ)^(β)], where P isthe probability to observe no crystallization event within time t, τ theaverage induction time. The stretched exponential exponent β served as ameasure for the spread of time scales characterizing the nucleationprocess, or the distribution of kinetic barriers arisen from theheterogeniety of the system. Such heterogeniety can be attributed to theheterogeneity of the polymer microstructure identified using SANS, whichresulted from a distribution of nucleation sites arising from spatialvariations in both the mesh size and chemical composition of thehydrogel at nanometer length scales. Note that β varies with the averagemesh size of the microgel. Polymer mesh size can impact the nucleationkinetics and an optimum average mesh size can be determined whichcorresponds to the fastest nucleation rate. At the optimum average meshsize, β is found to be the highest in both the cases of ASA and ACM(Table 5, M=400 g/mol; Table 6, M=200 g/mol). This is probably because,out of various types of nucleation sites in the microgel, the one withthe optimum mesh size and conformation is dominant in quantity andactivity, such that the majority of nucleation events take place at thistype of nucleation site, leading to a narrower distribution ofnucleation time scales. Taking this senario to extreme, β shouldapproach unity when the activity of a single type of nucleation site isso high that other nucleation sites are inactive by comparison.

Modification of PEGDA microgels with AM resulted in much fasternucleation kinetics of ASA overall. The nucleation induction timedistributions were better described by two-exponential models (Table 7,FIG. 24) instead of the stretched exponentials obtained with PEGDAmicrogels (Table 5). Two exponential processes yielded two distinct timescales, τ₁ and τ₂, with τ₁ an order of magnitude faster than τ₂. Boththe two exponential processes were much faster than those obtained withPEGDA microgels, indicating that strong polymer-solute interactions ledto overall success of polymer gels in promoting nucleation. Two timescales possibly result from the presence of two dominant types of activenucleation sites on PEGDA-co-AM microgels. Recalling the hypothesizedpolymer microstructure as determined by SANS (FIG. 21), it is likelythat the segregation of AM functional monomers into regions of highlocal acrylate density results in two largely different types of activesites for nucleation. One type, in the acrylate-lean (and thus AM-lean)regions of the gel, are such that interactions between the solute andPEG subchain dominate the nucleation process. The other, in the acrylateand AM-rich regions of the gel, are such that interactions between thesolute and AM dominate the nucleation process. The latter AM-richdomains may serve as the more active nucleation sites due to favorablesolute-AM interactions (as characterized by higher partition coefficientand adsorption enthalpy), which correspond to the shorter averageinduction time of ASA, and the vise-versa for the AM-lean domains. Thisinterpretation is also consistent with the observation that the shortertime scale τ₁ is much less sensitive to the variation in the PEGmolecular weight M than T₂, the longer time scale (Table 7), since theAM-rich domain should be less affected by variation in the PEG subchainlength than the AM-lean domain. In the case of PEGDA microgels, althoughthere also exists structural heterogeneity due to microphase separationbetween acrylate-rich and acrylate-lean domains, such dramatic split ofnucleation times scales was not observed, probably because only theacrylate-lean domains are nucleation active given that ASA mainlyinteracts with the PEG subchain in PEGDA, as discussed earlier.

Similarly, nucleation of ACM in the presence of PEGDA-co-AM microgelssplit into two exponential processes as well, probably for the samereasons discussed above. In contrast to the observations from ASAsystems, the slower time scale τ₂, possibly associated with the PEGrich, AM lean nucleation sites, was not reduced from those obtained withPEGDA microgels, although the faster time scale τ₁ was shortened by atleast an order of magnitude as in the case of ASA. This observationindicates that modification of PEGDA with AM promoted nucleation of ACMin terms of the overall effect, however, to a lesser extent comparedwith the ASA system. The data also suggest that the AM-rich nucleationsites are much more active than the AM-lean ones, evidenced by the twoorders of magnitude difference between T₁ and T₂. However, suchdifferencewas not reflected in the partitioning results, where nosignificant improvement in the partition coefficients was seen afterchemical modification. Others factors such as the templating effect mayplay a more important role in this case, which are discussed later.

TABLE 5 Average nucleation induction times of ASA with the presence ofPEGDA microgels. M (g/mol) Bulk/130 200 400 575 700 τ (min) Notdetectable 1052 66.7 3500 210000 B NA 0.52 0.69 0.52 0.36 R² NA 0.990.96 0.96 0.92

Supersaturation S=2.1. Detailed experimental conditions were describedelsewhere.⁹ Induction time distribution data were fitted with stretchedexponentials via nonlinear least square regression: P=exp[−(t/τ)^(β)],where P is the probability to observe no crystallization event withintime t. The R² value corresponding to PEG₇₀₀DA samples is lower sincemuch fewer samples crystallized within the experimental time frame.

TABLE 6 Average nucleation induction times of ACM with the presence ofPEGDA microgels. M (g/mol) Bulk 130 200 400 700 τ (min) 37000 1600 4805300 37000 B 0.50 0.54 0.72 0.50 0.50 R² 0.97 0.96 0.96 0.97 0.97

Induction time distribution data were fitted with stretched exponentialsvia nonlinear least square regression: P=exp[−(t/τ)^(β)].

TABLE 7 Average nucleation induction times of ASA with the presence ofPEGDA-co-AM microgels. M (g/mol) 130 200 400 575 700 τ1 (min) 170 21 3951 33 τ2 (min) 4900 99 400 470 720 A 0.52 0.05 0.62 0.79 0.68 R² 0.980.99 0.99 0.99 0.99

Bulk solution is at the same crystallization condition as that withPEGDA microgels. Induction time distribution data were fitted with twoexponentials via nonlinear least square regression:P=a×exp(−t/τ₁)+(1−a)×exp(−t/τ₂).

TABLE 8 Average nucleation induction times of ACM with the presence ofPEGDA-co-AM microgels. M (g/mol) 130 200 400 τ1 (min) 55 88 70 τ2 (min)1360 12400 35000 a 0.23 0.36 0.29 R² 0.91 0.96 0.97

Bulk solution is at the same crystallization condition as that withPEGDA microgels. Induction time distribution data were fitted with twoexponentials via nonlinear least square regression:P=a×exp(−t/τ₁)+(1−a)×exp(−t/τ₂).

In FIG. 24: Effect of PEGDA-co-AM microgels on nucleation induction timestatistics of ASA. P is the probability for no nucleation event to occurwithin time t; FIG. 24A Effect of polymer mesh sizes on nucleationkinetics. Fitted parameters following the two-exponential model arelisted in Table 7. Data for M_(n)=575 and 700 g/mol are shown separatelyfor clarity; FIG. 24B and FIG. 24C Comparison of two exponential vs.stretched exponential models using PEG₅₇₅DA-co-AM (FIG. 24B) andPEG₇₀₀DA-co-AM (FIG. 24C) as representative examples.

Several effects may have contributed to the observed enhancement innucleation kinetics with chemically modified polymer gels. First,preferential partitioning can increase solute concentration in the gel.Particularly, given the adsorptive partitioning mechanism discussedearlier, the solute molecules are likely to be enriched around thepolymer matrix. The resultant increase in local concentration mayenhance effective solute-solute interactions. Higher soluteconcentration can lead to higher supersaturation in the gel, and hencelarger driving force for nucleation. However, this is not necessarilythe case, as described herein. Supersaturation (S) is the chemicalpotential difference (Δμ) in relationship Δμ=μ_(s)−μ_(c)=kTlnS, whereμ_(s) and μ_(c) are chemical potentials of solute in the solution or gelphase and in the crystal phase. Since the gel and the solution are atequilibrium, the solute molecules possess the same chemical potential inthe two phases, and therefore the supersaturation is not different inthe gel from that in solution. Although the thermodynamic driving forcemay not increase due to the presence of the polymer, the polymer maystill serve as a catalyst, which reduces the kinetic barrier tonucleation by concentrating the solute molecules to facilitate molecularcluster formation. PEGDA-co-AM gels were more effective than PEGDA inpromoting ASA nucleation, which can be partially credited to higheraverage solute concentration in the gel (FIG. 22), especiallyconsidering that the concentration in local domains may be even higherdue to chemical heterogeneity of the gel, as discussed earlier. As forthe ACM system, the average solute concentration increased onlymarginally in the modified gel (FIG. 22), and as such its contributionto overall nucleation expedition is less significant than in the case ofASA. However, it is still remarkable that by incorporating AM into thePEGDA matrix, a fast nucleation process was created with averageinduction times orders of magnitude shorter than those obtained withPEGDA alone (Table 8). This phenomenon may not be attributed solely tothe concentration effect at the chemical heterogeneity may polarize thesolute concentration between the AM-rich and AM-lean segments but theextent of concentration polarization should be small, based on the factthat the partitioning coefficient did not increase much after replacing50 v % of PEGDA with AM. Other contributing factors may include thedifference in specific polymer-solute interactions (templating effect),or the nanoscale structural heterogeneity of the polymer gel. Thetemplating effect by studying preferred crystal orientation on PEGDA andPEGDA-co-AM polymer films via X-ray diffraction was investigated.

Templating Effect of the Polymer Gel on Nucleation:

The templating effect may affect crystal nucleation by aligning thesolute molecules along the polymer chain via specific polymer-soluteinteractions. To capture specific polymer-solute interactions in asolvent environment, the crystal facets preferentially grown from apolymer surface in the solvent of interest were determined and thecomplimentary functional group interactions were inferred by inspectingthe molecular structures of surfaces in contact. Smooth and flat polymerfilms were prepared following the same formulation as used in themicrogel synthesis, except that no porogen and solvent were added to thepre-polymer mixture so as to minimize the variation in polymer meshsizes, allowing the focus to be on the polymer chemistry effect.

Shown in FIG. 25A, PEGDA films preferentially templated the growth of(002) plane of ASA, and PEGDA-co-AM the (011) plane, judging from therelative peak intensities in the XRD patterns compared with those of thebulk crystals. This result was verified by the observations under theoptical microscope that ASA crystals with elongated plate-like shapeslay on their sides on the PEGDA surface via the (002) planes (FIGS. 25Dand 25E), and stood tilted on the PEGDA-co-AM film via the (011) plane(FIG. 25C). Comparing the molecular structures of (002) and (011)planes, it can be deduced that the methyl and phenyl groups of ASAdominating the (002) plane mainly interact with the PEGDA polymer, andthe carboxyl group characteristic of the (011) plane may be responsiblefor interacting with the AM segments of PEGDA-co-AM. Such complimentaryinteractions between PEGDA and ASA are possible, because the phenyl andmethyl hydrogens of ASA, being next to electron-withdrawing groups, haveincreased tendency to interact with the oxygen of PEGDA. This type ofC—H . . . O interactions, though much weaker than primary hydrogenbonding, is abundant in many crystal systems, such as the aspirincrystal in which the methyl hydrogen interacts with the carbonyl oxygenin the ester group to form a dimer-like supermolecular synthon. However,one might expect that the carboxyl group of ASA should primarilyinteract with PEGDA via hydrogen bonding instead of phenyl and methylgroups. This scenario is not observed probably because the ASA carboxylgroup is well solvated by ethanol and water, and as such its interactionwith PEGDA is hindered. Compared with PEGDA, the AM segments in thePEGDA-co-AM polymer carry higher density of stronger hydrogen bondacceptors in amide moieties, which may be more effective in completingwith ethanol and water to form hydrogen bonds with ASA carboxyl groups.To summarize, the observed preferred crystal orientation induced byspecific polymer-solute interactions provides strong evidence for thetemplating effect of the polymer film on nucleation. ASA interacts withPEGDA via weak C—H . . . O interactions, whereas its interaction withPEGDA-co-AM is much stronger, possibly via hydrogen bonds formed betweenASA and AM. This result is consistent with the observed higher ASApartitioning in PEGDA-co-AM, and stronger binding between the two asmeasured by the ASA adsorption enthalpy on the polymer. Given strongerinteractions with one end of the ASA molecule, AM was found to be moreeffective in aligning ASA molecules along the polymer chain, and therebylowered the entropic penalty during nucleus formation, leading tofurther shortened induction times.

In FIG. 25: Preferred orientation of ASA crystals on polymer films;(FIG. 25A) Comparison of XRD patterns of ASA crystals grew from PEGDAand PEGDA-co-AM polymer films to that of bulk crystals. The results arenot sensitive to variation in M_(n) and representative patterns areshown. (002) peak is separated from the (011) peak by a 2θ angle of 0.17degree (calculated from Cambridge Structure Database). The two peaks canbe unambiguously identified given that the resolution of XRD measurementis 0.02°; (FIGS. 25B-25E) Optical images of ASA crystals nucleated frombulk (FIG. 25B), the PEGDA-co-AM surface (FIG. 25C), and the PEGDAsurface (FIGS. 25D-25E); Scale bar is the same for all images; (FIGS.25F-25G) Molecular structures of (002) and (011) facets of ASA crystal.The dotted line indicates the top surface of the corresponding facet.

Similarly, preferred orientation of ACM crystals on polymer films wasalso observed, which further verifies the existence of templating effectimposed by the polymer network. XRD study showed that PEGDA inducedgrowth of (011) and its higher index plane (022) almost exclusively,while PEGDA-co-AM preferentially templated (101) and its higher indexplane (202) as well as (111) (FIG. 26A). It is evident from the opticalimages (FIGS. 26B-26E) that the prism-shaped ACM crystals exhibitedrandom orientations when crystallized from bulk, and seemed to assumecertain through-plane orientations when nucleated on the respectivefilms, judging from similar crystal morphology from the top view. Seenfrom molecular structures of templated crystal facets (FIGS. 26F-26H),all planes present phenolic hydroxyl groups to the surface, on the otherhand, (101) and (111) planes are different in chemistry from (011) inthat they better expose the amide group, although the difference is notas apparent as that between (002) and (011) of ASA. Such differenceimplies that after introducing AM into the PEGDA network, the polymerstrengthens its interactions with ACM by forming hydrogen bonds with theamide group of ACM, in addition to with the phenolic hydroxyl group.These observations help explain the moderate increase in partitioncoefficients after gel modification. Interestingly, both the amide andphenolic hydroxyl groups that AM preferentially interacts with are alsoused for forming the ACM crystal structure (Form I), which isessentially a network of intermolecular hydrogen bonding between the twogroups. This may suggest that, with the ability to hydrogen bond withboth the groups in the solvent of interest, the AM segment could actlike a ‘catalyst’ for crystal nucleation by facilitating hydrogen bondformation among the aligned ACM molecules, and lead to a fast nucleationprocess observed in the induction time study with modified gels (Table8).

In FIG. 26: Preferred orientation of ACM crystals on polymer films;(FIG. 26A) Comparison of XRD patterns of ACM crystals grew from PEGDAand PEGDA-co-AM polymer films to that of bulk crystals. All ACM crystalsare form I, the monoclinic form. (FIGS. 26B-26D) Optical images of ACMcrystals nucleated from bulk (FIG. 26B), the PEGDA surface (FIG. 26C),and the PEGDA-co-AM surface (FIG. 26D). Scale bar is the same for allimages; (FIG. 26E) ACM molecular structure. The functional group (i)preferentially interacts with PEGDA, (ii) with AM, and (iii) interactswith both PEGDA and AM. (FIGS. 26F-26H) Molecular structures of (011),(022), (111) and (101) facets of ASA crystal. Above the dotted line isthe top surface of the corresponding facet.

In conclusion, the role of polymer-solute interactions in controllingsolute nucleation was demonstrated by tuning the chemical composition ofthe polymer microgels used for inducing nucleation. When AM co-monomerwas introduced into the PEGDA matrix via co-polymerization, ASAnucleation kinetics was promoted by up to four orders of magnitude,while nucleation of ACM was also enhanced by up to two orders ofmagnitude. Comparing the ASA and ACM systems, the extent of nucleationacceleration generally correlated with the strength of polymer-soluteinteractions as characterized by solute partition coefficients andadsorption enthalpy. The effect of polymer-solute interactions onnucleation further manifested in the split of nucleation time scales dueto the presence of nucleation sites of distinct chemical compositions inthe microgels, inferred from SANS data. Favorable polymer-soluteinteractions promoted nucleation by two means. First, it led to highersolute concentration in the gel, which enhanced the effectivesolute-solute interactions. Second, specific polymer soluteinteractions, as evidenced by the preferred crystal orientation onpolymers, facilitated molecular alignment along the polymer chain.

Experimental Section

Materials:

Poly(ethylene glycol) diacrylate with average molecular weights ofM=200, 400, 575, and 700 g/mol and tri(ethylene glycol) diacrylate(M=130 g/mol), 4-acryloyl morpholine, poly(ethylene glycol) with M=200g/mol (PEG₂₀₀)_(,) 2-hydroxy-2-methyl-1-phenyl-propan-1-one (DC1173)photoinitiator, Tween20 non-ionic surfactant, and ethanol (99.9%) werepurchased from Sigma Aldrich Chemical Co. and used as received with nofurther purification. Deionized water (18.3 MΩ) was obtained using aMillipore MilliQ purification system. For PEGDA microgel pre-cursors,solutions containing 25% PEG_(M)DA, 25% PEG₂₀₀, and 5% DC1173 by volumein ethanol were prepared for each of the values of the molecular weightM used. Similarly, for PEGDA-co-AM microgels, solutions containing 15%PEG_(M)DA, 15% AM, 25% PEG₂₀₀, and 5% DC1173 photoinitiator by volume inethanol were prepared for each of the values of the molecular weight Mused. Aspirin (99%) was purchased from Alfa Aesar and acetaminophen(99.0%) from Sigma Aldrich, both used with no further purification.Perdeuterated ethanol (d-ethanol, 99.9%), was purchased from CambridgeIsotope Laboratories, and used without further purification.

Microgel Synthesis:

Cuboid microgel particles were synthesized by stop flow lithography(SFL). Microfluidic channels with straight, rectangular cross-section(width=300 μm, height=30 μm) were prepared by soft lithography. Briefly,polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) was poured on anSU-8 photoresist patterned silicon wafer and cured to create abas-relief microchannel device. Channels with end reservoirs were cutfrom the wafer with a scalpel and inlet and outlet ports were punchedinto the device with a blunt syringe (Small Parts, Inc.) to introducethe hydrogel pre-cursors. A photomask featured with square shapes wasdesigned using AUTOCAD and printed at 50,800 dpi by FineLine Imaging(Colorado Springs, Colo.). For SFL, the microfluidic device was placedon a translating stage inverted microscope. The inlet channel was loadedwith a hydrogel pre-cursor using a pressure-controlled manifold. Themask was placed in the field-stop of the microscope and square featureswere projected to the pre-cursor by ultraviolet (UV) exposure from aLumen 200 lamp (Prior) through a wide excitation UV filter set (11000v2:UV, Chroma) while the flow of pre-cursor was stopped. The ultimatefeature sizes of the patterned squares were 30 μm×30 μm, determinedthrough fluorescence imaging of the microchannel during UV illumination.Pulses of UV exposure were obtained by a computer-aided UV shutter(UniBlitz). Incident UV intensities were measured using a UVA Power andDose meter (ACCU-CAL-30 UVA, DYMAX). In all experiments, the measuredexposure was 0.89 μW, and the UV exposure time was fixed at 200 msec.Particles were collected through the outlet channel into amicrocentrifuge tube reservoir containing 0.2% v/v Tween20 in a mixtureof 62/38 water/ethanol (v/v). Tween20 was added to the outlet reservoirin order to render the microgels colloidally stable during purification.

SFL was performed until approximately 50,000 particles were synthesized.The reservoir tube containing particles was then removed from themicrofluidic device. The tube was placed in a minicentrifuge (GalaxyMiniStar, VWR Scientific) at 6000 rpm for 8 seconds in order to sedimentthe microgels. The supernatant was removed, and the particles werere-suspended in 1 mL of a rinsing fluid and vortex mixed for 10 seconds.This procedure was repeated several times in order to eliminate anyremaining unreacted pre-polymer solution. The first 3 washes wereperformed using 62/38 water/ethanol (v/v) with 0.2% Tween20, and 3 finalwashes were performed using 62/38 water/ethanol (v/v) with no Tween20 toeliminate excess surfactant.

Equilibrium Swelling Measurements:

Equilibrium swelling measurements were carried out as follows. Briefly,it was assumed that the as-synthesized dimensions of the microgelswithin the preparative microfluidic device were in the “relaxed” state(corresponding to a θ-solvent for the polymer), and their side length,L₀, was measured in situ. After purification and transfer into theappropriate crystallization solvent, the swollen side length, L,dimensions of the microgels were measured using DIC microscopy. Thesemeasurements were used to obtain the volumetric swelling ratio,R=(L/L₀)³, assuming isotropic swelling of the microgel. Finally, theapparent average mesh size ξ was estimated using Flory-Rehner theory,which gives the average PEG_(M)DA molecular weight between cross-links,M _(c), as

$\begin{matrix}{\frac{1}{{\overset{\_}{M}}_{c}} = {\frac{2}{M} - \frac{{\ln \left( {1 - {R\; \varphi_{p,0}}} \right)} + {R\; \varphi_{p,0}} + {\chi \left( {R\; \varphi_{p,0}} \right)}^{2}}{\varphi_{p,0}\rho_{p}{{\underset{\_}{V}}_{s}\left( {R^{1/3} - \frac{R}{2}} \right)}}}} & (2)\end{matrix}$

where χ is the Flory chi parameter, ρ_(p) is the density of the polymer,and V_(s) is the molar volume of the solvent. The quantity φ_(p,0) isthe volume fraction of polymer in the microgel, where it is assume thatthe polymerization proceeds to completion, and thus φ_(p,0) is equal tothe volume fraction of monomers in the hydrogel pre-cursor.Subsequently, the apparent, average mesh size of the hydrogel, ξ, isgiven by

$\begin{matrix}{\xi = {R\; {\varphi_{p,0}^{1/3}\left( \frac{2C{\overset{\_}{M}}_{c}}{{\overset{\_}{M}}_{n}} \right)}^{1/2}l}} & (3)\end{matrix}$

where C is the characteristic ratio and l is the average bond length ofthe polymer. Estimations of ξ were carried out using model parametersfor PEGDA in a water-ethanol. It was assumed that these model parameterswere unchanged by either the PEG_(M)DA molecular weight or the presenceof the co-monomer AM. The latter is a particularly significantapproximation, and will be evaluated subsequently.

Small Angle Neutron Scattering (SANS):

SANS was performed at the National Institute of Standards and TechnologyCenter for Neutron Research (NCNR). Samples were prepared by loadinghydrogel pre-cursors (with the compositions previously described) forthe PEG₂₀₀DA, PEG₇₀₀DA, PEG₂₀₀DA-co-AM, and PEG₇₀₀DA-co-AM microgelsinto standard titanium scattering cells with a path length of 1 mm. Inorder to polymerize the material, samples were irradiated with ahandheld UV lamp with an output intensity of 0.2 mW/cm² for 1 minute,resulting in a total UV dose which is approximately equivalent to thatsupplied during SFL of microgel particles.

SANS measurements were carried out on the NG7 30 m SANS instrument withthe 10CB sample environment. Temperature control was obtained using aJulaba temperature bath unit at 25° C., and samples were left toequilibrate for at least 30 min prior to measurement. Scattering usingincident neutrons of wavelength λ=6 Å and a wavelength spread (FWHM) ofΔλ/λ=11% was collected at detector distances of 1 m with 20 cm offset, 4m, and 13.5 m for high-q measurements. Scattering using incidentneutrons of wavelength λ=8.09 Å and a wavelength spread (FWHM) ofΔλ/λ=11% was collected at a detector distances of 15.3 m for low-qmeasurements. USANS measurements were performed on the BT5 perfectcrystal diffractometer within the 6CB sample environment. Temperaturecontrol was obtained using a Julaba temperature bath unit, and sampleswere left to equilibrate for at least 30 min prior to measurement. Datawere reduced using NIST IGOR software package in order to obtain theabsolute scattered intensity, I(q). The incoherent background intensity,I_(bk), was determined using a Porod analysis of the data at highq-values.

Partition Coefficient Measurements:

Partition coefficients of ASA in PEGDA-co-AM gels from its bulk solutionwere determined as follows. In brief, a series of gels with varying meshsizes of approximately 5 mm in diameter and 0.5 mm in thickness weresynthesized via UV polymerization following the same formula as used inthe microgel synthesis. The residue solvent, porogen and monomermolecules were removed by extensive washing with solvent ethanol andsubsequent vacuum drying. The dry gels were then immersed in excessivevolume of 38 mg/ml ASA solution in 38/62 (v/v) ethanol/water at 15° C.for overnight. After equilibrium swelling was reached, the swollen gelswere pad dried and dropped into excessive volume of water to releaseASA. The total mass of ASA released was determined by measuring theequilibrium concentration of its degradation product in water, salicylicacid (SA), with UV-Vis spectroscopy, after ASA aqueous solution was agedfor a week to achieve complete hydrolysis. The ASA partition coefficientwas calculated as the ratio of ASA mass fraction in solution absorbed bythe gel to that in bulk solution. Partition coefficient of ACM wasdetermined by the same method. The gels were immersed in 95 mg/ml ACMsolution at 8° C. instead. Since ACM is stable in water, itsconcentration was determined directly after the swollen gel was immersedin water for 24 hours. Three to four independent repeats were carriedout for each type of sample to obtain the standard error of thepartition coefficient.

Isothermal Titration Calorimetry (ITC):

ITC measurements were performed on a TA Instruments NanoITC calorimeter.All experiments were performed at 23° C. using injections of ΔV=10 μL oftitrant, with a waiting time of 1000 sec in between injections and 25injections per measurement. For all measurements, The differential heatinput, q(t), was measured as a function of time t over all injections,followed by integration of q(t) over each individual injection to obtainthe molar heat of injection, Q(T,P,c). The molar heat of injection canthen be cumulatively added over all previous injections, yielding thetotal molar heat, Q_(tot) (T,P,c).

The primary measurement involves titration of a solution containingc_(inj)=21 mg/mL ASA in 38/62 (v/v) ethanol/water (loaded in theinjection syringe) into a suspension containing microgel particles at aconcentration of 1 particle/μL in 38/62 (v/v) ethanol/water. For thisprocess, the molar heat of injection contains several contributions

Q(T,P,c)=c _(inj) ΔV(ΔH _(ASA-gel)(T,P,c)+ΔH _(dil) ^(ASA)(T,P,c)+ΔH_(dil) ^(gel)(T,P,c))  (4)

where ΔH_(ASA-gel) is the molar enthalpy of interaction between ASA andthe microgel particles, and ΔH_(dil) ^(i) is the molar enthalpy ofdilution of component i (ASA or gel, respectively) in 38/62 (v/v)ethanol/water. In order to determine ΔH_(ASA-gel), independentmeasurements of the ΔH_(dil) ^(ASA) and ΔH_(dil) ^(gel) were made byperforming measurements where 21 mg/mL ASA in 38/62 (v/v) ethanol/waterwas injected into a sample containing only 38/62 (v/v) ethanol/waterwithout particles, and where 38/62 (v/v) ethanol/water without ASA wasinjected into a 1 particle/O_, suspension 38/62 (v/v) ethanol/water.Subsequently, eq. (X) was used to subtract the measured dilutionenthalpies from the initial measurements of Q(T,P,c) in order to obtainΔH_(ASA-gel). Subsequently, the total, cumulative enthalpy evolved overall injections due to polymer-solute interactions, ΔH_(ASA-gel) ^(tot),is calculated by summing the instantaneous enthalpy of interaction,ΔH_(ASA-gel), over all injections:

$\begin{matrix}{{\Delta \; {H_{{ASA} - {gel}}^{tot}\left( {T,P,c} \right)}} = {\sum\limits_{c_{j} = 0}^{c}{\Delta \; {H_{{ASA} - {gel}}\left( {T,P,c_{j}} \right)}}}} & (5)\end{matrix}$

where c_(j) is the concentration of the j^(th) injection.

Nucleation Induction Time Measurement:

Crystallization of ASA from 38/62 (v/v) ethanol/water mixture in thepresence of PEGDA-co-AM microgels of various mesh sizes were conductedin an RS 10 Clarity Solubility Station (Thermo Fisher Scientific).Around 500 microgels were dispersed in every 1 ml of 38 mg/ml ASAsolutions in 38/62 (v/v) ethanol/water mixture, and kept suspended bystirring the solution at 700 rpm. 10 such samples were loaded into theClarity station at once and quench cooled to 15° C. to generate asupersaturation of 2.1. The onset of crystallization was signaled by thesudden drop of IR transmission signal through the solution. The timetaken from the moment the desired supersaturation was achieved to themoment the IR signal dropped was the nucleation induction time. 10samples were cycled 5 to 10 times to yield the induction timeprobability distribution. Experimental conditions were kept the same forsamples with PEGDA gels and those with PEGDA-co-AM gels for directcomparison. During the experiments, the solution was inspected under theoptical microscope at intervals to make sure the microgels were neitheraggregated nor dgraded. For ACM, same procedures were followed with 95mg/ml ACM solution in 38/62 (v/v) ethanol/water cooled to 8° C. toachieve a supersaturation of 3.7.

Preferred Crystal Orientation Via XRD:

Polymer films of various PEG molecular weights were synthesized via UVpolymerization using pre-polymer mixtures of the same formulations asused for microgel synthesis, but without adding solvent ethanol andporogen PEG200. 30 μl pre-polymer mixture was sandwiched between a glassslide and a quartz slide, both 75 mm×25 mm in size. The glass slide wassilanized with vinyl trichlorosilane, which co-polymerizes with themonomer to graft the polymer film to the glass substrate via covalentbonds. The quartz slide was used as a template to make polymer filmswith the minimum surface roughness possible. The sandwiched pre-polymermixture was subjected to 70 mW/cm² UV light for 5 min to complete thepolymerization, with the whole sample area irradiated fairly uniformlyin the 5000-EC UV Curing Flood Lamp (Dymax Corporation). The quartzslide was subsequently lifted to leave the flat and smooth polymer filmconformed to the glass substrate. After synthesis, the polymer filmswere immersed vertically in 25 mg/ml ASA solution in 38/62 (v/v)ethanol/water mixture, which was filtered with 0.45 μm PTFE membranesyringe filter before adding the polymer films. The solution was thensealed and cooled from 25° C. to 3° C., and visually inspected everyhour. Once crystals were spotted, the polymer film was withdrawn fromthe solution to terminate crystallization and immediately dipped intoD.I. water tank vertically to remove loosely attached crystals from bulk(ASA is essentially insoluble in water at 3° C.). The backside of theglass substrate was used as a control to determine if all loose crystalswere removed from the polymer film. Bulk crystals were obtained at thesame condition and serves as the control sample for XRD analysis. ForACM system, same procedure was carried out with 80 mg/ml ACM solution in38/62 (v/v) ethanol/water mixture.

The specific crystal planes grown from the polymer film was analyzedusing PANalytical X'Pert PRO Theta/Theta Powder X-Ray Diffraction Systemwith Cu tube and X'Celerator high-speed detector. 20 mm×20 mm samplearea was irradiated by the X-ray in one scan using programmabledivergence slit with 20 mm irradiated length and 20 mm mask to ensureenough crystals on the polymer film were sampled to yield thestatistically representative preferred orientation. Three scans wereperformed with one polymer film to cover almost the entire surface area.Since only the diffraction from the crystal plane parallel to thepolymer film surface was seen by the X-ray detector, the peak that wassignificantly more intense relative to that of bulk crystals correspondsto the preferred nucleation face.

Example 5

The following example describes non-limiting embodiments relating tocrystallization of polymorphs at confined interfaces.

Introduction:

Controlling polymorphism, the ability of a compound to self-assembleinto multiple crystal structures, has been a long-standing challenge invarious fields of application. In particular, for pharmaceuticalsystems, polymorphs often exhibit distinct physical properties, whichhave profound impact on drug bioavailability, stability, processibility,etc. Both nucleation and crystal growth, two steps constituting acrystallization process, were shown to affect polymorphic outcomes. Thelack of understanding and control of nucleation, however, remains as amajor roadblock in current polymorphism research. One of the mostchallenging, yet less-explored aspects in controlling nucleation ofpolymorphs is to decipher the role of interfaces in the nucleationprocess, since in practice almost all nucleation events occurheterogeneously, a.k.a, at an foreign interface. Designed nucleationsubstrates can be very useful in controlling polymorphism. For instance,some molecular compounds tend to crystallize in multiple polymorphsconcomitantly under the same condition, which could be caused byassorted unknown nucleation sites in the solution. By ‘seeding’ thesolution with designed nucleation ‘catalyst’ to selectively lower thenucleation barrier of a particular polymorph, heterogeneous nucleationinduced by unintended contaminants can be avoided and controlledpolymorph nucleation can be achieved.

Several types of substrates have been studied for screening orcontrolling polymorphs of molecular crystals, including crystallinesubstrates, 2D ordered surfaces such as self-assembled monolayers, andinsoluble polymer surfaces. On these flat and smooth substrates,polymorph selectivity seems to be best achieved when both latticematching (epitaxy) and complimentary chemical interactions at thecrystal-substrate interface are satisfied. In recent years, materialsimposing a nanoscopically confined environment for crystallization havealso been explored for polymorph control, such as controlled pore glasswith pores ranging from a few to a hundred nanometers, andmicroemulsions with drop sizes of 2-10 nanometers. Stabilization ofmetastable polymorphs in nanoconfinement sufficiently small was oftenobserved. To explain these observations, evidence was presented that thelarge surface area to volume ratio can alter the relative polymorphstability. Another hypothesis frequently evoked states that when thepore size becomes smaller than the critical nucleus size of a polymorph,its crystallization was hindered in confinement. However, thesearguments fail to account for the nucleation-templating effect ofconfinement interfaces. Moreover, the kinetic aspect of polymorphcontrol under nanoconfinement has been ignored, which is particularlyglaring given the definitive role of nucleation kinetics in affectingpolymorphic outcomes. In fact, systematic studies on the kinetics ofpolymorph nucleation have been scarcely reported in general, not only inthe nanoconfinement literature.

This example describes the of a novel material, polymer microgels, forunderstanding and controlling polymorph crystallization of molecularcompounds in a confined environment. The microgels exhibited a mesh-likestructure, formed by crosslinking polyethylene glycol diacrylate(PEG_(M)DA) of various PEG subchain molecular weight M (g/mol). Whenimmersed in solution, the microgel swelled by uptaking solute andsolvent molecules owing to favorable interactions, and the degree ofswelling, which varied as a function of the PEG subchain length, definedits average mesh size, a quantity typically used for describing themicrostructure of the swollen polymer network. With mesh sizes rangingfrom a few angstroms to several nanometers, the polymer networkpartitioned the absorbed solution and restricted the mobility ofadsorbed solute molecules, as such providing a confined environment forcrystallization to take place. Using polymer microgels of tunable meshsizes, the nanoconfinement effect on polymorphism was investigated usingtwo model compounds, carbamazepine (CBZ) and5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (ROY). Theirpolymorphic outcomes were strongly dependent on the polymer mesh sizeand chemical composition. In addition, there exhibited an evidentcorrelation between the nucleation kinetics and the polymorphic outcome.The underlying mechanism was examined from three aspects: the influenceof mesh size, preferential partitioning, and specific polymer-soluteinteractions. The selectivity of polymorph nucleation may arise from thetemplating effect driven by specific polymer-solute interactions, which,facilitated with an optimum spatial configuration imposed by theconfinement effect, may enhance the nucleation of a particular polymorphto the greatest extent.

Results and Discussion:

Monodispersed cubelike PEG_(M)DA microgels (FIG. 27 left), with Mranging from 130 to 700 g/mol were synthesized by Stop-Flow Lithography,The mesh sizes vary from 0.7 to 1.5 nm in solvent ethanol (Table 9),estimated from equilibrium swelling by the Flory-Rehner theory. Theaccuracy of this method for obtaining mesh sizes has been confirmed withSmall-Angle Neutron Scattering in a different solvent. The microgelswere utilized for controlling polymorph crystallization by suspending˜10 μg of microgels per 1 ml solution by stirring. Such a low microgelconcentration may be sufficient to effect drastic change in thecrystallization behavior (discussed later), underscoring theeffectiveness of polymer microgels in controlling crystallization. Inall crystallization experiments, supersaturation of the solution wasgenerated by cooling instead of solvent evaporation, despite itspopularity in numerous polymorph studies, to yield better control overthe crystallization process.

CBZ and ROY were selected as model compounds to represent both packingpolymorphism (CBZ) and conformational polymorphism (ROY), where CBZpolymorphs have the same conformer arranged in differing molecularpacking motifs, and ROY, in comparison, assumes distinctive molecularconformations in various packing arrangements, altering its conjugationstate and thus the color among different polymorphs. Both molecules havebeen studied extensively for purposes of polymorph screening andcontrol. CBZ possesses four known anhydrous forms, and ROY has ten knownforms with seven structurally characterized. The complexity of the twosystems poses challenges for their polymorph control. Specifically,concomitant crystallization (simultaneous crystallization of multipleforms in the same liquid), has been reported for both the systems. Inaddition, crystallization of ROY polymorphs also suffers from poorreproducibility, owing to the stochastic nature of its polymorphcrystallization.

Crystallization of CBZ Polymorphs Induced by Microgels:

Out of the four known anhydrous forms of CBZ, namely, Triclinic form I,Trigonal form II, Primitive monoclinic form III, and C-centeredmonoclinic form IV, form III is the most stable under ambientconditions, followed by form I, IV and II, with the energy separationbetween form III and II less than 0.7 kcal/mol. Such a narrow energywindow suggests the sensitivity of CBZ crystallization to experimentalparameters. There have been some inconsistencies in previous reports onthe CBZ polymorphic outcome during crystallization from solution undersimilar conditions. For instance, when crystallized from highlysupersaturated ethanol solution (often with supersaturation S >3) bycooling to low temperatures (T<10° C.), various forms have beenobtained. At a lower supersaturation (S=2) and higher temperature (T=25°C.), concomitant crystallization of forms II and III has been observedalso from ethanol solution. In this example, CBZ crystallization wasfound to be quite sensitive to experimental conditions such as solidimpurity concentration, solution water content (trace amount), andstirring speed etc., which could explain some of the aforementionedinconsistencies (e.g., see Methods section). Therefore, allcrystallization conditions were strictly controlled to yieldreproducible results.

At the experimental conditions employed (S=1.63, T=25° C., 2 ml purifiedsolution stirred at 300 rpm), concomitant crystallization of Forms I andII was consistently observed (˜100 trials) when crystallized from thebulk of ethanol solution (Table 9), both polymorphs with needle-likecrystal habits. Occasionally, pure Form III was also obtained from bulkexperiments. The possibility of observing both I and II forms due tosolvent-mediated polymorph transformation was eliminated because thepolymorphic composition did not exhibit statically significant changeduring aging of the crystal mixtures in solution from the onset ofnucleation, which were sampled at intervals and characterized by XRD.Interestingly, when PEG_(M)DA microgels with M≧400 g/mol were added intothe solution, pure Form II was crystallized (˜250 trials) as identifiedby XRD, whose needle-shaped crystals were observed to grow from themicrogel surface (FIG. 27 right). However, such polymorph selectivitycould not be attained using microgels with M<400 g/mol (˜100 trials)where the polymorphic outcome was quite similar to that of the bulksamples (Table 9), only with a decreased mass fraction of Form I in themixture obtained (FIG. 28 discussed later) and a lower frequency ofoccurrence of Form III.

In FIG. 27: Optical micrographs of PEG₄₀₀DA microgels as synthesized(left) and CBZ form II needles grown on PEG₄₀₀DA (right), in which threemicrogels covered with CBZ needles are indicated with arrows, and thecontour of the middle one is traced with lines to delineate the cubicgel.

TABLE 9 Effect of PEG_(M)DA microgels on the average nucleationinduction times and polymorphic outcomes of CBZ. M (g/mol) Bulk 130 200400 575 700 ξ (nm) NA 0.7 0.8 1.1 1.3 1.5 Polymorph II & I, occasionallyII & I II & I II II II III τ (min) 427 ± 13 222 ± 5 174 ± 11 10.9 ± 33 ±1 49 ± 1 0.3 β 0.86 0.99 0.73 1.00 0.61 0.94 R² 0.986 0.982 0.960 0.9760.981 0.987Average mesh size ξ was estimated from equilibrium swelling experimentsin solvent ethanol. Polymorphic outcomes were analysized by XRD, andcorresponding polymorphic compositions were shown in FIG. 28. Inductiontime distribution data were fitted with stretched exponentials vianonlinear least square regression: P=exp[−(t/τ)^(β)], where P is theprobability to observe no crystallization event within time t. X-rayDiffraction patterns of CBZ were obtained from bulk solution and in thepresence of PEG_(M)DA microgels with M=200 and 575 g/mol. CBZ forms Iand II peaks were observed. A peak at 9.00° appeared in some patterns,corresponding to CBZ dihydrate which forms during filtration, especiallywhen the ambient humidity is high.

Nucleation induction time statistics of CBZ were determined with orwithout the presence of PEGDA microgels, wherein P is the probabilityfor observing no crystallization event within time t. Stretchedexponential model was employed to fit the data (see Table 9).

Accompanying the impact on CBZ polymorphic outcomes is the ability ofthe microgels in altering the CBZ nucleation kinetics, characterized bythe average nucleation induction time τ (Table 9). Induction time wasmeasured by monitoring IR transmission signal passing through a solutionin controlled temperature environment and stiffing condition. Oncenucleation occurred, the solution became turbid in seconds indicated bya sharp drop of IR signal, due to secondary nucleation and rapid crystalgrowth. The statistical nature of nucleation necessitated a large numberof experiments (50-100) for obtaining the distribution of inductiontimes, from which the average induction time τ was regressed with astretched-exponential model, P(t)=exp[−(t/τ)^(β)], where P is theprobability to observe no crystallization event within time t. In brief,the stretched-exponential model modified the single exponential modelderived from the Poisson statistics, P(t)=exp (−t/τ) by adding anexponent β to the dimensionless induction time t/τ to capture the spreadin characteristic time scales caused by a distribution of nucleationactive sites present in the system.

Shown in Table 9, microgels with M=130 and 200 g/mol effectivelyshortened the CBZ average induction time by 2-3 fold relative to that ofthe bulk samples, whereas at least an order of magnitude reduction wasobserved with microgels of higher M. More importantly, there exhibited astrong correlation between the extent of nucleation acceleration and thepolymorph selectivity, wherein Form II was exclusively obtained onlywith the microgels sufficiently effective in promoting nucleation.Considering that Form II is the least stable polymorph at ambientconditions, the observed polymorph selectivity towards a higher energyform may be driven by kinetic factors, in which case, the presence ofmicrogels preferentially lowered the kinetic barrier to Form IInucleation, as opposed to switching the relative stability between FormI and II.

Mechanistic Investigations into CBZ Polymorph Selectivity:

The microgels could alter the relative nucleation rates of CBZpolymorphs through various means. First, the concentration effect wasinvestigated, based on the knowledge that the microgels have the abilityto concentrate solute molecules via favorable polymer-soluteinteractions. Equilibrium partitioning experiments revealed that CBZconcentration in the polymer gel was 3-4 times as high as that in thebulk (see Table 13). In addition, a generally higher partitioncoefficient κ in microgels with larger M suggests that CBZ preferredinteracting with PEG to acrylate segments. To assess the effect ofsolute concentration on polymorphic outcomes, bulk crystallizationexperiments were conducted at a series of starting concentrations andthe resultant Form I and II mixtures were analyzed with XRD to quantifythe polymorph compositions. Shown in FIG. 28, increasing soluteconcentration reduced the Form I mass fraction and thus biased thepolymorphic outcome towards the less stable Form II. This trend was notunexpected since in practice less stable forms are typically generatedby increasing the supersaturations to drive the system towards kineticcontrol regime. However, using this strategy Form I could not beeliminated to obtain pure Form II, even at concentration as high as 140mg/ml (S=6.7). In fact, when concentration increased beyond 60 mg/ml,the polymorph composition became irreproducible since before thesolution was cooled to desired supersaturation level, crystallizationalready ensued. In contrast, the microgels can take the system into aparameter space inaccessible through conventional means. For sampleswith microgels, the same trend was observed as with the bulk samples(FIG. 28), which indicated higher solute concentration in the gel couldfacilitate selective nucleation of Form II. The high degree ofselectivity cannot be explained quantitatively with only theconcentration effect, and thus other contributing factors were examined,such as the nucleation templating effect of the PEGDA polymer.

Partition coefficients (κ) of CBZ in PEG_(M)DA microgels from ethanolsolutions were determined, wherein κ is defined as the ratio of solutemass fraction in solution confined in the gel to that in the bulk.

In FIG. 28: Effect of solute concentration on the polymorphiccomposition of CBZ crystals. For samples with PEG_(M)DA microgels,X-axis corresponds to the effective solute concentration of solutioninside the gel, calculated by multiplying the solute partitioncoefficient (see Table 13) with the bulk concentration, 34 mg/ml for allsamples with microgels. The X error bars are from partition coefficientmeasurements, and the Y error bars calculated from XRD measurements onthree independent samples. The mass fraction of Form I, η, wascalculated following

${\eta = {k\frac{I\left( \theta_{I} \right)}{{I\left( \theta_{I} \right)} + {I\left( \theta_{II} \right)}}}},$

where I denotes relative peak intensity. θ_(I) and θ_(II) are thecharacteristic peak positions (2θ) for Forms I and II, respectively. Inthis case, θ_(I)=12.345° and θ_(II)=5.046°. Coefficient k,experimentally determined, converts the peak intensity fraction to thepolymorph mass fraction (see Methods section).

Besides increasing the solute concentration in the gel, favorablepolymer-solute interactions can also induce a templating effect, bywhich it directs the CBZ molecules towards a particular orientation viamolecular recognition events and thereby reducing the entropic costduring nucleus formation. This microscopic phenomenon can be expressedmacroscopically as preferred orientation of crystals on flat polymersurfaces, which can be detected via XRD. The PEGDA polymer surface onlyinduced nucleation of a particular set of crystal planes of Form II,i.e., (110) and its higher index planes, irrespective of the polymermesh size or the PEG subchain molecular weight (FIG. 31A). Specificityas high as such suggests that the templating effect may be a substantialfactor in microgel-induced polymorph selectivity. To identify thespecific polymer-CBZ interaction responsible for directing Form IInucleation, the surface chemistry of II (110) and II (440) were comparedagainst other major crystal facets not nucleated from the polymersurfaces, namely, II (410), I (022) and I (024) (Note that the XRDpatterns were obtained with randomly oriented crystal powders, andtherefore capture a statistical average of all crystal facets grown fromthe system). All II (410), I (022) and I (024) facets exhibited similarsurface chemistries, dominated by the phenyl group on the azepine ringand decorated with carboxamide group (FIG. 31D; only I (022) is shown),which left the vinyl group a distinctive functionality characterizingthe II (440) facet. This analysis implied that it is the vinyl group ofCBZ mainly engaged in its interaction with PEGDA (FIG. 31E), possibly byforming the C—H . . . O between the CBZ vinylic hydrogen of CBZ and theoxygen of the PEG subchain. Albeit weak, such interactions were found toplay an important role in directing nucleation process and indistinguishing polymorphs of many organic crystals.

In summary, both the concentration effect and the templating effect arefound to contribute to the observed CBZ polymorph selectivity induced bypolymer gels. Considering the striking similarity of the intermolecularinteractions between Form I and II of CBZ, the polymorph selectivityachieved with the microgels is significant. It is also worth noting thatthe CBZ polymorphic outcomes were sensitive to the polymer mesh sizes,with exclusive nucleation of Form II only obtained using microgels oflarger mesh sizes, the implication of which is discussed later andsummarized as the mesh size effect.

In FIG. 29: Specific CBZ-polymer interactions inferred from preferredcrystal orientations. (FIG. 29A) XRD pattern of CBZ crystals grown onPEG_(M)DA films. Relative peak intensities were found to be independentof M. A representative pattern is shown. (FIG. 29B, FIG. 29C) Surfacestructures of Form II facets preferentially nucleated on polymersurfaces. (FIG. 29D) Surface structure of a facet characteristic of FormI not grown from polymer surface. (FIG. 29E) Functionality inferred topreferentially interact with PEGDA polymer (colored blue).

Crystallization of ROY Polymorphs Induced by Microgels:

ROY crystallization is well known for its poor polymorph selectivity fortwo reasons. First, when crystallized from solution, multiple polymorphscan be obtained (often in pure forms, occasionally concomitant) from thesame solution under seemingly identical condition. Second, duringcrystallization from supercooled melt, concomitant polymorphs arefrequently observed controlled by both the nucleation and crystal growthkinetics. During solution crystallization, the poor selectivity mayarise from a broad distribution of molecular conformations in solution,and/or the small free energy difference between ROY polymorphs as in thecase of CBZ.

In FIG. 30: Optical micrographs of ROY crystallized from (FIGS. 30A-30C)bulk and on (FIGS. 30D-30I) microgels, specifically, with M=400, 575,700, 400, 400, 400 g/mol in images FIGS. 30D, 30E, 30F, 30G, 30H, 30Irespectively. Y, R and ON denote yellow prism, red prism and orangeneedle forms. Scale bars for images (FIGS. 30D-30I) are the same asshown in (FIG. 30F). In images (FIGS. 30D, 30E, 30G, 30H, 30I), thecubic microgels are buried with tiny ROY crystals grown from theirsurfaces, whereas in image (FIG. 30F), only one large single crystalnucleated on the gel, leaving the red-colored microgel clearly visible.The originally transparent microgel became red in solution due to highpreferential partitioning of ROY into the gel (see Table 13) orpolymer-solute interaction induced conformation change.

In each experiment, crystallization of pure forms of either Y (yellowprisms), R (red prisms) or ON (orange needles) were observed fromethanol solution stochastically (FIG. 30), and their frequencies ofoccurrence were calculated by conducting 50-150 experiments in each case(FIG. 31). Nucleation induction time data from bulk solution or withPEG₁₃₀DA microgels were fitted with the stretched exponential model andthe average induction time thus regressed represents an average of allforms (Table 10), since the frequencies of occurrence for R and ON weretoo low for separate statistical analysis. Low values of stretchedexponential exponent β (Table 10) indicate a wide spread of nucleationtimes scales, possibly associated with a broad conformation distributionof ROY in solution since β as low as such has not been observed withsystems exhibiting packing polymorphism.²² Induction time data obtainedwith microgels of M=200-700 g/mol can be described withmulti-exponential models (Table 11), with each exponential decay processcorresponding to each particular form, and its characteristic time scalethe average nucleation induction time of each form. In themulti-exponential model, each exponential decay process may be betterrepresented with a stretched exponential, however, a simple exponentialsuffices in this case and decent fit was obtained with R² very close tounity (Table 11). This may be due to the time scales corresponding todifferent polymorphs are at least an order of magnitude apart and thus,the spread of times scales within each decay process, described by thestretched exponential exponent β, becomes secondary in comparison.

For bulk crystallization without microgels, predominant Form Y at thechosen experimental condition (see Methods section) was observed,occasionally Form R and ON (FIGS. 30A-30C, FIG. 31 left). The relativestability of the three observed polymorphs are Y>ON>R at this condition.This observation is consistent with previous reports, for instance,primarily Y was obtained from various supercooled solutions. Theaddition of microgels had significant impact on ROY nucleation kineticsand polymorph frequency of occurrence, the extent of which variedconsiderably with the polymer mesh size. Shown in Table 10, PEG₁₃₀DAmicrogels expedited nucleation of Y form, however, its effect oncrystallization of other forms was not very pronounced. When PEG₂₀₀DAmicrogels were injected into the ROY solution, nucleation of Y wasfurther accelerated, all of which occurred within 100 min, and Form Ralso started appearing at a detectable rate. The observed promotion of Yand R nucleation kinetics was reflected in increased frequencies ofoccurrence for both the forms (FIG. 31 left). With addition of PEGDAgels of larger mesh sizes (Table 11), Form ON nucleation was acceleratedinto the detectable range as well, but its average rate was much slowerthan those of Forms Y and R in all cases. Meanwhile, nucleation of FormR continued to be promoted, until a maximum was reached with PEG₅₇₅DAmicrogels, at which point the average induction time of R was reduced to200 min from well over 10000 min without microgels. Correspondingly, thefrequency of occurrence for R also peaked at M=575 g/mol, replacing Y tobecome the dominant polymorph (FIG. 31 left). Evidence of ROY polymorphsnucleated on microgels was shown in FIGS. 30D-30I.

Previous experiments have found that the frequency of occurrence of FormR has been quite low compared with Y, ON during solution crystallizationfrom various solvents, particularly when supersaturation was achieved bycooling as did in this example. While other methods have been used forscreening rare ROY polymorphs, the reproducibility and selectivity werenot reported. In this example, the addition of polymer microgelsimproved the Form R frequency of occurrence by up to 20 times even atrelatively low supersaturation, which is not attainable by conventionalmeans.

Nucleation induction time statistics of ROY with or without the presenceof PEGDA microgels were determined. For each type of samples, 50-100experiments were performed to obtain the induction time statisticswherein P is the probability for observing no crystallization eventwithin time t is estimated from the fraction of samples haven'tcrystallized at this time point. Either stretched exponential model ormulti-exponential models were employed to fit the data (see Tables 10and 11).

TABLE 10 Average nucleation induction times of ROY in bulk and withPEG₁₃₀DA microgels. Samples τ, min β R² Bulk 10000 ± 2000 0.37 ± 0.020.97 PEG₁₃₀DA 4000 ± 750 0.25 ± 0.01 0.97

Induction time distribution data were fitted with stretched exponentialsvia nonlinear least square regression: P=exp[−(t/τ)^(β)], where P is theprobability to observe no crystallization event within time t. τ is anaverage of induction times for all Y, R, ON forms.

TABLE 11 Average nucleation induction times of ROY with PEG_(M)DAmicrogels, (M = 200-700 g/mol). Microgels τ(Y), min τ(R), min τ(O), mina b R² PEG₂₀₀DA 26.0 ± 1.5  9000 ± 6000 NA 0.550 NA 0.983 PEG₄₀₀DA 22.0± 0.6 1600 ± 400 3300 ± 1200 0.298 0.330 0.995 PEG₅₇₅DA 10.0 ± 0.8 200 ±20 2900 ± 500  0.205 0.350 0.996 PEG₇₀₀DA 12.0 ± 1.2  2600 ± 400* 0.375NA 0.967 Induction time distribution data were fitted with superpositionof two or three exponentials via nonlinear least square regression.Two-exponential fit was employed for PEG₂₀₀DA and PEG₇₀₀DA samples: P =a · exp[−t/τ(Y)] + (1 − a) · exp[−t/τ(R)], with τ(R) an average of formsR and O for the PEG₇₀₀DA sample. Three-exponential fit was used forPEG₄₀₀DA and PEG₅₇₅DA samples: P = a · exp[−t/τ(Y)] + b · exp[−t/τ(R)] +(1 − a − b) · exp[−t/τ(O)], where Y, R, and O represent the yellow, redand orange needle forms respectively. *τ, an average induction time ofForm R and ON, given the lack of data points to distinguish the two.

In FIG. 31: Polymorph frequency of occurrence in 12 mg/ml ROY-ethanolsolution with or without microgels of various mesh sizes (left) and athigher solution concentrations, C₀ (right). Frequency of occurrence iscalculated as the percentage of samples crystallized in a particularform within 1440 min out of the total number of samples. The analysisfor 12 mg/ml solution is carried out previously. In summary, addition ofPEGDA microgels accelerated crystallization of all three forms, Y, R andON. Particularly, nucleation of a metastable form R was preferentiallyinduced as to become the dominant form at an optimum mesh size M=575g/mol, whereas Y crystallized almost exclusively without any microgels.Nucleation of another metastable form ON was also promoted when M>200g/mol, however, to a much less extent compared with R. Interestingly,ROY nucleation kinetics is extraordinarily sensitive to the variation inmesh sizes, particularly Form R.

Mechanistic Investigations into ROY Polymorph Selectivity:

The impact of microgels on ROY polymorphic outcome can be comprehendedfrom the perspectives of the concentration effect, the templatingeffect, and the mesh size effect Similar to CBZ, ROY also exhibitspreferential partitioning in the gel phase (see Table 13), which leadsto concentrations 6-11 times as high as that of the bulk solution. Thisresult is visibly reflected in the red color of microgels (FIG. 30F)imparted from highly concentrated ROY. Besides, the fact that κ is muchhigher at larger M indicates ROY predominantly interacts with the PEGsubchain. The influence of higher solute concentration on polymorphicoutcomes was investigated with bulk crystallization experiments (FIG. 31right). ON became the dominant form with the increase of concentration,which is in accordance with previous reports. This concentration effectmay explain accelerated nucleation of ON when microgels with M=400-700g/mol were added (FIG. 31 left), considering that the ROY concentrationin these microgels is much higher than in others (see Table 13). Theconcentration effect may not fully account for the observation thatcrystallization of R form was particularly promoted by microgels in thisembodiment. The strong dependence of R form nucleation kinetics andfrequency of occurrence on M may suggest that the mesh size effect andthe ROY-polymer interactions may play a key role in controlling R formnucleation.

The ROY-polymer interactions were probed via preferred crystalorientations on flat PEG_(M)DA films. From the bulk solution, ROYcrystallized in Form Y predominantly. In comparison, PEG_(M)DA filmstemplated nucleation of R, as well as Y, which is consistent with theobservations with microgels. In particular, a few crystal facets showmuch stronger XRD peak intensities compared with the bulk sample, i.e.,Y (020), R (111) and R (220), indicating that they are the dominantcrystal facets nucleated on the polymer. To verify this observation, thecontribution from each crystal facet was quantified and listed theprominent ones in Table 12. The preferred crystal orientation variedsignificantly as a function of M, the PEG subchain molecular weight.Polymers with M=130, 200 g/mol favor R (111) and R (220). As Mincreases, percentage of R decreases and Y increases to become thedominant polymorph on M=575, 700 g/mol, which is contact with thepolymer via Y (020) and/or (040) facet. This observation may suggestthat the acrylate group of PEGDA could be responsible for templating R,whereas the PEG subchain induced nucleation of Y. The polymorphcrystallization was sensitive to polymer-solute interactions.

XRD patterns of ROY crystallized from bulk solution and on PEGDA filmsunder the substantially similar conditions were obtained. Additionalpeaks observed from crystals templated by polymer films but not frombulk crystals were observed. Reference patterns were calculated from CSDusing POWD-12++.

TABLE 12 Percentages of ROY crystals in various orientations (hkl) onPEG_(M)DA polymer films. M (g/mol) Y (020), % Y (040), % R (111), % R(220), % 130 7.5 3.4 38.0 46.8 200 3.5 1.6 36.2 53.6 400 23.6 9.9 28.824.9 575 26.9 13.1 12.9 20.0 700 50.9 16.9 9.8 14.4

Analysis on the XRD data is carried out by normalizing the measured peakintensities I_(p) ^(i) with the reference peak intensities I_(bulk) ^(i)from the bulk sample, following the formula

${\eta_{i} = {\frac{I_{p}^{i}/I_{bulk}^{i}}{\sum\limits_{i}\left( {I_{p}^{i}/I_{bulk}^{i}} \right)} \times 100}},$

where η_(i) is the percentage of crystals in orientation i, and p isshort for polymer. Minor orientations (<10% on all films) are consideredbut not shown in the table. Percentages are highlighted as bold fordominant orientations in each case.

TABLE 13 M (g/mol) 130 200 400 575 700 CBZ 3.0 ± 0.4 2.7 ± 0.1  3.8 ±0.2  3.8 ± 0.04  3.7 ± 0.1 ROY 6.3 ± 0.2 6.1 ± 0.9 11.0 ± 0.4 11.3 ±1.6  11.8 ± 1.1

Partition coefficients (κ) of CBZ and ROY in PEG_(M)DA microgels fromethanol solutions. κ is defined as the ratio of solute mass fraction insolution confined in the gel to that in the bulk. The error bars arecalculated from three to four independent repeats. To study the specificpolymer-solute interactions, the molecular structures of preferentiallynucleated crystal facets was examined as shown in FIG. 32. R (111) and R(220) exhibited very similar surface functionalities. The cyano andnitro moieties, although exposed to the surface, were engaged in ROYself-interactions, leaving the amine and methyl groups more available tointeract with the polymer surface (FIG. 32D), probably with the acrylategroup as inferred from the XRD results (Table 12). Specifically, theamine hydrogen of ROY can form hydrogen bond with the carbonyl ofacrylate group, and the methyl group of ROY can interact with acrylateby forming C—H . . . O contact, which is often referred to as asecondary hydrogen bond As for Form Y, either Y (020) or Y (040) or bothwere possibly templated by the polymer, which cannot be distinguishedfrom the XRD results since they are parallel planes. However, it isunlikely for Y (040) would associate with PEGDA because it resembles Y(022) and Y (042) in surface chemistry (not shown), which are notfavored by the polymer but are among the main facets of bulk crystals.All Y (022), (042) and (040) facets are featured with cyano and/or nitromoieties, which are more likely to engage in ROY intermolecularinteractions or solvated by ethanol. Therefore, the phenolic hydrogenexposed to the Y (020) surface can be inferred to be responsible forinteracting with the PEG subchain (FIG. 32D), probably by forming C—H .. . O contact with the ethylene oxide oxygen.

The identified specific ROY-polymer interactions may help elucidate therole of templating effect in polymorph selection. By interacting withthe phenolic hydrogen of ROY, the PEG subchain may cause two effects.First, it can help align the ROY molecules in a particular orientation,and as such better exposes other moieties in ROY for self-interactionsand facilitates molecular clusters formation, which is a key step tonucleation. Second, it can hinder the phenolic hydrogen-nitrorecognition essential to R polymorph formation (not found in other formswith known structures except for OPR), resulting in preferentialnucleation of Y polymorph on polymer films with higher M. Likewise, byhydrogen bonding with the amine moiety of ROY, the acrylate group of thepolymer can interfere with the amine-cyano hydrogen bond unique to Ypolymorph (not found in other forms), and as such facilitatescrystallization of R polymorph.

In FIG. 32: Specific polymer-ROY interactions inferred from preferredcrystal orientations. (FIGS. 32A-32C) Molecular structures of ROYcrystal facets preferentially grown from the polymer surface. The solidline indicates the top surface of the corresponding facet. R and Ydenote red and yellow ROY polymorphs. Prominent intermolecularinteractions in ROY crystals are denoted with green dotted lines if theinteraction is between the two in-plane molecules as depicted, and withdotted lines if it is between one in-plane molecule and another moleculein the next layer in through-plane direction. (FIG. 32D) Molecularstructures of monomers of ROY and PEGDA. ROY functional groups coloredblue are inferred to preferentially interact with PEG subchain, andthose colored red with the acrylate group.

Molecular recognition motifs in ROY crystals of forms Y (FIGS. 32A-32B)and R (FIGS. 32C-32D) were prepared and both intermolecular interactionsand intramolecular interactions were determined. Other supermolecularrings may form by different combinations of the same set ofintermolecular interactions.

Following the above discussions, the templating effect of the microgelsalone is may bias the polymorph selectivity towards R at lower M and Yat higher M. Observed continuous nucleation acceleration of Form Y withan increase in M (Tables 10 and 11), however, the trend of R may beinfluenced by more than the templating effect and the concentrationeffect Regarding the mesh size effect, the existence of an optimum meshsize for expediting nucleation of R, at which point, its frequency ofoccurrence was kinetically driven to a maximum. The optimum mesh sizemay arise from the interplay between polymer-solute interactions andspatial confinement imposed by the polymer mesh. Specifically, when themesh size is too small, most of solute molecules are adsorbed to thetightly intertwined polymer chain given the large volume fraction ofpolymers, thereby reducing the molecular mobility and hinderingeffective solute-solute interactions essential to nucleus formation(FIG. 33A); when the mesh size is too large, a smaller fraction ofsolute is associated with the polymer and the nucleation-templatingeffect of the polymer due to specific polymer-solute interactions ismuch less significant (FIG. 33C); at the optimum mesh size, thepolymer-solute and solute-solute interactions are balanced, enabling thesolute molecules aligned by adjacent polymer chains to act concertedlyin forming the nucleus given appropriate spacing (FIG. 33B). Thishypothesis implies that the optimum mesh size may not be the same forpolymorphs of the same compound, since their nucleation events aretemplated by different specific polymer-solute interactions as discussedabove, and the spacing required for molecular cooperativity may differas well. For ROY system, the separation of optimum mesh sizes between Yand R is central to the observed favorable polymorph selectivity towardsR at M=575 g/mol, which overcomes the opposite trend of templatingeffect that favors Y with increasing M. As for the CBZ system, the meshsize effect is also evident (Table 9). There exhibited an optimum meshsize at M=400 g/mol corresponding to the fastest nucleation rate of FormII, whose mass fraction concurrently attained maximum. The polymorphselectivity maintained at 100% even when M increased beyond optimum,probably because the mesh size effect was counterbalanced by thetemplating and concentration effect, which favored Form II at higher M.

In FIG. 33: Schematic illustrating the mesh size effect on nucleation.Lines denote the polymer mesh with crosslinking points indicated usingsmall circles. The polymer mesh drawn here not necessarily representsthe actual physical model, but is sufficient to illustrate the role ofvarying confinement size. Solute molecules are signified withellipsoids, whose one end is colored darker grey and preferentiallyinteracts with the polymer chain, and the other end color coloredlighter grey responsible for self-interactions. One example of suchmolecules is CBZ, with the darker grey end corresponding to the vinylgroup on the azepine ring that interacts with the PEG subchain, and thelighter grey end corresponding to the carboxamide group, which dimerizesin CBZ crystals. The molar ratios of solute to monomer unitsconstituting the polymer are drawn to scale, which are calculated fromCBZ partitioning experiments. The relative size of the solute to themesh size is also drawn to scale approximately for CBZ system. Therelative fraction of solute molecules adsorbed to the polymer chain isestimated by assuming that the number of solute binding sites scaleslinearly with the PEG subchain length. The optimum mesh size for CBZnucleation was found to be 1.1 m (Table 9). Therefore the nucleusformation is illustrated in (FIG. 33B) as highlighted with yellowbackground.

In conclusion, the polymer microgels were demonstrated as materials forcontrolling crystallization of polymorphs. PEGDA microgels selectivelyinduced nucleation of Form II of CBZ, while concomitant crystallizationof Form I and II were observed from bulk. In another example, themicrogels improved ROY polymorph selectivity towards Form R by up to 20times, whereas bulk crystallization predominantly produced Y or ONdepending on the supersaturation. Through these examples, the polymergels showed ability to take the polymorphic system into occurrencedomains not accessible through conventional methods. Furthermore,through these mechanistic investigations, the nucleation-templatingeffect and the spatial confinement imposed by the polymer network wereimportant in achieving polymorph selectivity. With this insight,selective crystallization of a desired polymorph can be achieved bydesigning the polymer chemistry and microstructure.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed: 1-6. (canceled)
 7. A method of making a pharmaceuticalproduct, comprising: crystallizing a pharmaceutically active agent on atleast one surface of or associated with at least one excipient, whereinthe at least one excipient promotes crystallization of thepharmaceutically active agent and the at least one excipient comprisesan imprinted surface having a plurality of surface features selected tobe complimentary to a known shape and/or angle(s) of a selected knowncrystal structure of the pharmaceutically active agent; and forming apharmaceutically acceptable composition comprising the pharmaceuticallyactive agent and the at least one excipient wherein the process is freeor essentially free of mechanical steps for combining thepharmaceutically active agent with the at least one excipient. 8-11.(canceled)
 12. The method of claim 7, wherein the pharmaceuticallyactive agent is aspirin or acetaminophen. 13-18. (canceled)
 19. Themethod of claim 7, wherein the at least one excipient comprises apolymeric material.
 20. The method of claim 19, wherein the polymericmaterial is a hydrogel.
 21. The method of claim 20, wherein thepolymeric material is formed using UV polymerization.
 22. The method ofclaim 20, wherein the polymeric material is formed by reacting at leastone monomer and a crosslinking agent using UV irradiation. 23-24.(canceled)
 25. The method of claim 7, wherein the plurality of surfacefeatures comprises a plurality of wells.
 26. The method of claim 7,wherein the shape of the surface features are triangles, squares,rectangles, trapezoids, pentagons, hexagons, or octagons.
 27. The methodof claim 7, wherein the surface features have an average cross sectionof greater than about 10 nm.
 28. The method of claim 7, wherein thesurface features have an average cross section between about 10 nm andabout 1000 nm.
 29. The method of claim 28, wherein the surface featureshave an average depth of less than about 10 mm.
 30. The method of claim7, wherein the surface features have an average depth of between about50 nm and about 1 nm. 31-33. (canceled)
 34. The method of claim 7,wherein a portion of the surface comprises a plurality of functionalgroups complimentary to a plurality of functional groups of thepharmaceutically active agent.
 35. (canceled)
 36. The method of claim34, wherein the surface comprising the plurality of functional groups isthe surface comprising the plurality of surface features.
 37. (canceled)38. The method of claim 34, wherein the plurality of functional groupscomprises a plurality of hydroxyl functional groups, a plurality ofcarboxylic acid ester functional groups, a plurality of nitrogencontaining base functional group, a plurality of aryl functional groups,a plurality of carboxyl functional group, a plurality of tertiary amidefunctional groups, or combinations thereof.
 39. The method of claim 7,wherein the surface features have an average cross section between about10 nm and about 500 nm.
 40. The method of claim 7, wherein the surfacefeatures have an average cross section between about 10 nm and about 100nm.
 41. The method of claim 28, wherein the surface features have anaverage depth of less than about 500 nm.
 42. The method of claim 28,wherein the surface features have an average depth of less than about 5nm.
 43. The method of claim 7, wherein the surface features have anaverage depth of between about 30 nm and about 1 nm.